pyrroloquinoline quinone is a plant growth …pyrroloquinoline quinone is a plant growth promotion...

Pyrroloquinoline Quinone Is a Plant Growth PromotionFactor Produced by Pseudomonas fluorescens B161

Okhee Choi, Jinwoo Kim, Jung-Gun Kim, Yeonhwa Jeong, Jae Sun Moon,Chang Seuk Park, and Ingyu Hwang*

Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul NationalUniversity, Seoul 151–921, Korea (O.C., J.K., J.-G.K., Y.J., I.H.); Division of Plant Resources and Environment,Gyeongsang National University, Jinju 660–701, Korea (O.C., C.S.P.); and Plant Genome Research Center, KoreaResearch Institute of Bioscience and Biotechnology, Daejeon 305–333, Korea (J.S.M.)

Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growthpromotion by this organism, we mutagenized wild-type strain B16 using VKm elements and isolated one mutant, K818, whichis defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutantK818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genesresponsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragmentidentified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinolinequinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato(Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruitnumber, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5to 1,000 nM of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings,confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant inplants.

Bacteria that colonize plant roots and enhance plantgrowth by any mechanism are referred to as plantgrowth-promoting rhizobacteria (PGPR). PGPR havebeen applied on various crops to enhance growth, seedemergence, crop yield, and disease control, and somehave been commercialized (Kloepper, 1992; Glick, 1995;Dey et al., 2004). The use of PGPR in sustainable agricul-ture is steadily increasing and offers an attractive way toreplace chemical fertilizers, pesticides, and supplements.

PGPR can promote plant growth indirectly or di-rectly. Indirect plant growth promotion is mediated byantibiotics or siderophores produced by PGPR thatdecrease or prevent the deleterious effects of plant-pathogenic microorganisms (Leong, 1986; Sivan and

Chet, 1992). Direct plant growth-promoting factorsinclude various phytohormones (Xie et al., 1996), sol-ubilization of soil phosphorus and iron (De Freitaset al., 1997), N2 fixation (Christiansen-Weneger, 1992),increases in nitrate uptake (Sophie et al., 2006), reduc-tion of membrane potential in roots (Bashan andLevanony, 1991), 1-aminocyclopropane-1-carboxylatedeaminase (which modulates plant growth and devel-opment; Safronova et al., 2006), and the production ofvolatiles as potential signal molecules (Ryu et al., 2003).

In mammals, pyrroloquinoline quinone (PQQ) func-tions as a potent growth factor, although its biologicalfunctions are not fully understood (Smidt et al., 1991;Steinberg et al., 1994). PQQ has attracted considerableinterest because of its presence in a wide variety offoods and its remarkable antioxidant properties (Smidtet al., 1991; Kumazawa et al., 1995; Mitchell et al., 1999;He et al., 2003). PQQ is found in plant and animaltissues in the nanogram-to-gram range even thoughplants and animals do not produce PQQ themselves(Kumazawa et al., 1992, 1995). PQQ is water soluble,heat stable, and has the ability to carry out redox cycles(Stites et al., 2000). It has been reported that PQQ acts asa reactive oxygen species (ROS) scavenger by directlyneutralizing reactive species in Escherichia coli (Misraet al., 2004). PQQ acts as a noncovalently bound redoxcofactor of several bacterial dehydrogenases, includingmethanol dehydrogenase and Glc dehydrogenase

1 This work was supported by the Crop Functional GenomicsCenter of the 21st Century Frontier R&D Program (grant no.CG2131), funded by the Ministry of Science and Technology of theRepublic of Korea, and by a Korea Research Foundation Grant,funded by the Korean Government (Ministry of Education andHuman Resources Development, Basic Research Promotion Fund;grant no. KRF–2006–005–J04701).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Ingyu Hwang ([email protected]).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.112748

Plant Physiology, February 2008, Vol. 146, pp. 657–668, www.plantphysiol.org � 2007 American Society of Plant Biologists 657

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(GDH; Duine et al., 1990; Stites et al., 2000). GDH is aquinoprotein that uses PQQ as a cofactor and is in-volved in the periplasmic oxidation of Glc to gluconicacid, resulting in the solubilization of the poorly solu-ble calcium phosphate (Babu-Khan et al., 1995). Inmammals, picomolar amounts of PQQ enhance DNAsynthesis activity in human fibroblasts and displaynerve growth factor-inducing activity (Naito et al.,1993; Yamaguchi et al., 1993). PQQ-deficient diets im-pair growth, cause immunological defects, and de-crease fertility in mice (Killgore et al., 1989; Steinberget al., 1994). Recently, PQQ has been proposed tofunction as a vitamin in mammals, following the iden-tification of the first potential eukaryotic PQQ-dependentenzyme (Kasahara and Kato, 2003). However, this ideais still controversial and it is unknown whether PQQaffects plant development and growth in vivo.

The biochemical pathways of PQQ biosynthesis arenot fully understood, but it is known that Glu and Tyrare precursors (Houck et al., 1991; Unkefer et al., 1995).Genes involved in PQQ biosynthesis have been iden-tified from various bacteria, including Acinetobactercalcoaceticus (Goosen et al., 1989), Methylobacteriumextorquens AM1 (Toyama et al., 1997), Klebsiella pneu-moniae (Meulenberg et al., 1992), Gluconobacter oxydans(Felder et al., 2000), and Pseudomonas fluorescens CHA0(Schnider et al., 1991). The pqqABCDEF genes areconserved in bacteria, but the biochemical functionsof the encoded proteins are largely unclear. Recently,PqqC has been reported to be the final catalyst in theproduction of PQQ (Magnusson et al., 2004).

We have studied the promotion of plant growth byP. fluorescens B16, which was isolated from the roots ofgraminaceous plants. The wild-type B16 colonizes theroots of various plants and produces an antibacterialcompound that is effective against plant root patho-gens, such as Agrobacterium tumefaciens and Ralstoniasolanacearum (Kang and Park, 1997; Kim et al., 1998;Kim et al., 2003). This organism also significantlypromotes the growth of cucumber (Cucumis sativus)and barley (Hordeum vulgare) under greenhouse andfield conditions (Kim et al., 1998). However, the mech-anism of plant growth promotion by this strain isunknown. In this study, we report that PQQ synthe-sized by P. fluorescens B16 is a key factor involved ingrowth promotion in tomato (Solanum lycopersicum),cucumber, Arabidopsis (Arabidopsis thaliana), and hotpepper (Capsicum annuum). Moreover, we report fourpreviously unidentified pqq genes and demonstratethat expression of the pqq genes is regulated by atranscriptional activator, PqqH. This article reportsthat PQQ promotes plant growth in vivo.

RESULTS

Isolation of a Plant Growth Promotion-Defective Mutant

Following random mutagenesis of P. fluorescens B16with VKm, the mutant K818 was isolated from 2,000prototrophic colonies due to its failure to promote the

growth of the tomato cultivar ‘Kwangsoo’ in a rock-wool system. Heights of the tomato plants were mea-sured every 3 d up to 27 d after the treatment of tomatoplants at the four- or five-leaf stage with K818 or wild-type B16. Mutant K818 failed to promote tomatogrowth, whereas the height of plants treated withB16 was increased by approximately 25% at 27 d aftertreatment (Fig. 1). To determine whether K818 wasable to colonize tomato roots, bacterial populations onthe roots were examined. The colonizing populationsof B16 and the mutant strain K818 on roots were 5.2 3105 and 5.3 3 105 colony-forming units (CFU) g21

roots, respectively, indicating that the plant growthpromotion-defective mutant K818 has the same root-colonizing activity as wild-type B16.

Identification of Genes Responsible for

Plant Growth Promotion

To identify genes that confer plant growth promo-tion in wild-type B16, the DNA region flanking theVKm insertion in the mutant K818 was isolated by selfligation of chromosomal DNA digested with EcoRI.The rescued plasmid pOK8 had an insert of 5.8 kb anda 2.8-kb PstI fragment from pOK8 was subcloned intopBluescriptII SK1, resulting in pOK12. Analysis of theDNA sequences of the flanking regions from pOK12revealed that the VKm element in the mutant K818was inserted in a gene homologous to a LysR-typetranscriptional regulator of P. fluorescens Pf0-1 (Tables Iand II; Fig. 2). The cosmid clone pOK40, spanning theflanking regions of the gene disrupted in K818, wasisolated from a genomic library of wild-type B16 by

Figure 1. Tomato plant growth promotion following treatment withwild-type P. fluorescens B16, the mutant K818, and K818 carryingpOK40. The height of the tomato plants was recorded at 3-d intervalsup to 31 d after inoculation. The values are means of three replicationsper experiment pooled from three experiments. Vertical bars indi-cate SDs.

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colony hybridization using the 1.8-kb HindIII/PstIfragment of pOK12 as a probe (Fig. 2). When pOK40was mobilized into the mutant K818, the plant growth-promoting effect was restored to that of wild-type B16(Fig. 1).

A restriction enzyme map of the 25-kb insert ofpOK40 was constructed (Fig. 2). To further character-ize the insert, we mutagenized pOK40 and pOK53,which carries a 13.4-kb BamHI fragment from pOK40in pLAFR3, with Tn3-gusA, followed by marker ex-change into wild-type B16. We identified 12 Tn3-gusAinsertions that interfere with plant growth promotion,delineating the essential region for this function (Fig.2). The 13.4-kb BamHI fragment from pOK40 wassubcloned into pBluescriptII SK1, resulting in pOK51,for DNA sequencing. The sequence of the fragmentcontains 11 potential open reading frames (ORFs),which were named pqqA, B, C, D, E, F, H, I, J, K, and M,as the Tn3-gusA insertions in each ORF abolished PQQ

production (Fig. 2). pqqA, B, C, D, E, F, and M genesexhibit strong similarity to those of the P. fluorescensstrain Pf0-1 (Table II; Fig. 3). Proteins PqqH and I of P.fluorescens B16 are highly similar to a LysR-type tran-scriptional regulator and a class-III aminotransferasepresent upstream of the pqq gene cluster in P. fluores-cens Pf0-1, respectively (Table II). pqqJ is predicted toencode a 13.6-kD protein exhibiting 53% identity and68% similarity to a putative cytoplasmic protein fromSalmonella enterica sp. enterica serovar Choleraesuis str.SC-B67 (Table II). PqqK is predicted to be a protein of11.6 kD that is similar to a DNA-binding protein ofSinorhizobium meliloti 1021 (Table II).

Expression of pqq Genes in a PqqH-Dependent Mannerin Nutrient-Limiting Conditions

To determine how the pqq genes of P. fluorescens B16are expressed, we analyzed their expression levels

Table I. Bacterial strains and plasmids

Ampr, Ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance; Nalr, nalidixic acid resistance; Rifr, rifampicin resistance;Smr, streptomycin resistance; Spr, spectinomycin resistance; Tetr, tetracycline resistance.

Strain or Plasmid Characteristics Source or Reference

E. coliDH5a F2 F80 dlacZDM15D(lacZYA-argF)U169 endA1 recA1 hsdR17 (rk2mk1)

deoR thi-1 supE44l2gyrA96 relA1Gibco BRL

S17-1 Tra1, recA, Spr Simon et al. (1983)C2110 polA, Nalr Stachel et al. (1985)HB101 F- mcrB mrr hsdS20(rB

2mB2) recA13 leuB6 ara-14 proAe lacY1 galK2

xyl-5 mtl-1 rpsL20(Smr) supE44l2

Gibco BRL

P. fluorescensB16 Wild-type, Rifr Kim et al. (2003)K818 B16TVKm This studyBK1 B16 pqqHTV This studyBK544/BKS544 B16 pqqATTn3-gusA544/BK1 pqqATTn3-gusA544 This studyBK4/BKS4 B16 pqqBTTn3-gusA4/BK1 pqqBTTn3-gusA4 This studyBK306/BKS306 B16 pqqCTTn3-gusA306/BK1 pqqCTTn3-gusA306 This studyBK433/BKS433 B16 pqqDTTn3-gusA433/BK1 pqqDTTn3-gusA433 This studyBK109/BKS109 B16 pqqETTn3-gusA109/BK1 pqqETTn3-gusA109 This studyBK96/BKS96 B16 pqqFTTn3-gusA96/BK1 pqqFTTn3-gusA96 This studyBK24/BKS24 B16 pqqITTn3-gusA24/BK1 pqqITTn3-gusA24 This studyBK316/BKS316 B16 pqqJTTn3-gusA316/BK1 pqqJTTn3-gusA316 This studyBK175/BKS175 B16 pqqKTTn3-gusA175/BK1 pqqKTTn3-gusA175 This studyBK117/BKS117 B16 pqqMTTn3-gusA117/BK1 pqqMTTn3-gusA117 This study

PlasmidspBluescriptII SK1 Cloning vehicle; phagemid, pUC derivative, Ampr StratagenepLAFR3 Tra2, Mob1, RK2 replicon, Tetr Staskawicz et al. (1987)pRK415 Mob1, lacZ, Tetr Keen et al. (1988)pHoKmGus Promoterless GUS gene, Kmr, Ampr Bonas et al. (1989)pSShe Cmr Stachel et al. (1985)pHP45V V cassette, Spr, Smr Prentki and Krisch (1984)pOK8 5.8-kb self-ligated EcoRI clone from K818 This studypOK12 2.8-kb PstI fragment from pOK8 cloned into pBluescriptII SK1 This studypOK40 27.4-kb DNA fragment from strain B16 cloned into pLAFR3 This studypOK51 13.4-kb BamHI fragment from pOK40 cloned into pBluescriptII SK1 This studypOK53 13.4-kb BamHI fragment from pOK40 cloned into pLAFR3 This studypOK59 2.0-kb BamHI-HindIII fragment including the pqqH region from

pOK58 cloned into pLAFR3This study

pOK67 14-kb HindIII fragment harboring a 2.0-kb V cassette in thePshAI site within pqqH in pOK40 cloned into pRK415

This study

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using pqqTTn3-gusA fusion mutants grown in Luria-Bertani (LB) medium or Agrobacterium minimal me-dium (AB). None of the pqq genes were expressed athigh levels in LB medium, but each was expressed morestrongly in AB medium (Table III). This result indicatesthat pqq genes are expressed only under nutrient-limiting conditions. Because PqqH shows similarity to

a transcriptional regulator, we evaluated whetherPqqH influences expression of the other pqq genes byconstructing Tn3-gusA fusions of each pqq gene in thepqqHTV mutant BK1. Expression levels of the pqqgenes were significantly lower in the pqqHTV mutantBK1 and were restored by providing pOK59 carryingpqqH in trans (Table III), indicating that PqqH is a

Table II. Annotation of P. fluorescens B16 genes encoding Pqq proteins

Genea No. of Amino

Acid ResiduesbPutative Function [Organism]

(GenBank Accession No.)BLAST E Value

pqqA 24 PQQ A biosynthesis protein [P. fluorescens](CAA60731)

6e-06

pqqB 303 PQQ biosynthesis protein PqqB [P. fluorescensPf0-1] (YP_350886)

4e-164

pqqC 250 PQQ biosynthesis protein PqqC [P. fluorescensPf0-1] (YP_350887)

2e-143

pqqD 91 PQQ biosynthesis protein PqqD [P. fluorescensPf0-1] (YP_350888)

1e-44

pqqE 425 PQQ biosynthesis protein PqqE [P. fluorescensPf0-1] (YP_350889)

0.0

pqqF 812 Peptidase M16A, coenzyme PQQ biosynthesisprotein PqqF [P. fluorescens Pf0-1] (YP_350884)

0.0

pqqH 306 Transcriptional regulator, LysR family [P. fluorescensPf0-1] (YP_350893)

2e-153

pqqI 427 Aminotransferase class-III [P. fluorescens Pf0-1](YP_350892)

0.0

pqqJ 119 Putative cytoplasmic protein [Salmonella entericasubsp. enterica serovar Choleraesuis str. SC-B67](YP_218683)

1e-26

pqqK 106 Probable DNA-binding protein [Sinorhizobiummeliloti 1021] (NP_436416)

4e-31

pqqM 607 Peptidases S9, prolyl oligopeptidase active siteregion [P. fluorescens Pf0-1] (YP_350890)

0.0

aUnified nomenclature for genes that encode P. fluorescens B16 Pqq proteins. bPutative function ofan individual gene product is predicted based on homology to proteins of known function in other PQQsynthesis pathways.

Figure 2. Organization of the pqqABCDEFHIJKM genes. White arrows indicate the positions and orientations of the PQQbiosynthesis genes. Vertical bars in the maps indicate the positions and orientations of the Tn3-gusA insertions, and the majorphenotypes of the mutants are represented below the restriction map. The vertical bar with a black circle indicates the position ofthe VKm insertion in mutant strain K818. The vertical bar with a black triangle indicates the position of the V cassette insertion.B, BamHI; E, EcoRI; H, HindIII.

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transcriptional activator in pqq gene expression. pqqAexpression level was higher than those of the other pqqgenes and was less affected by PqqH (Table III).

Promotion of Tomato Plant Growth in a Hydroponic

Culture System

Because wild-type B16 promoted the growth of to-mato plants in a rockwool system, we tested whether itcould promote tomato plant growth in a hydroponicculture system. In this system, wild-type B16 increasedplant height by 19.8% and flower number by 42%, asmeasured at 65 d after treatment (Fig. 4). In addition,treatment with B16 increased the fruit number and totalfruit weight after the final harvest by 41% and 36%,respectively (Fig. 4). As expected, the mutant K818failed to confer growth promotion and K818 carrying

pOK40 did promote growth (Fig. 4). We repeated thegrowth study of tomato plants in the hydroponicculture system twice over 2 years and observed verysimilar results; therefore, only 1 year of data is pre-sented in Figure 4. These results indicate that thegrowth promotion of tomato plants by wild-type B16can be achieved in a hydroponic culture system.

PQQ Is a Key Element for Plant Growth Promotion

Based on the fact that mutations in pqq genes abol-ished plant growth-promotion activity of wild-typeB16, we examined whether the strain produces PQQby analyzing culture supernatants using reverse-phase(RP)-HPLC. PQQ was detected as 5-acetonyl-PQQ bycomparison with the elution times of synthetic PQQ and5-acetonyl-PQQ from the RP-HPLC chromatograms

Figure 3. Comparison of the pqq gene clusters of P. fluorescens B16 with those from P. fluorescens Pf0-1, Klebsiellapneumoniae, Acinetobacter calcoaceticus, Gluconobacter oxydans ATCC9937, and Methylobacterium extorquens AM1.Positions and orientations of the pqq genes are indicated by white and colored arrows. The same colors represent homologousencoded proteins. The organization and size of the genes are depicted based on nucleotide sequence data from GenBank. Thefollowing genes were used: P. fluorescens Pf0-1 (GenBank accession no.CP000094), K. pneumoniae (X58778), A. calcoaceticus(P07778 to P07783), G. oxydans ATCC9937 (AJ277117), PqqAB of M. extorquens AM1 (L25889), PqqCD and PqqE of M.extorquens AM1 (U72662), and PqqFG of M. extorquens AM1 (L43135).

Table III. Expression of the pqqABCDEFHIJM genes in LB or AB medium

Bacterial cells were grown for 12 h. One unit of GUS was defined as 1 nmol of 4-methyllumbelliferone released per bacterium per minute. Allvalues are means 6 SD of values from triplicate experiments. –, Not determined.

Tn3-gusA Fusion

Specific Activity of GUS (10211 CFU21)

B16 (Wild Type) BK1 (B16 pqqHTV) BK1 (B16 pqqHTV, pOK59)

LB AB LB AB LB AB

pqqATTn3-gusA544 12.7 6 1.2 396.7 6 5.9 3.5 6 0.4 142.0 6 3.4 25.5 6 2.3 415.5 6 5.4pqqBTTn3-gusA4 1.6 6 0.6 25.9 6 2.0 1.0 6 0.2 0.2 6 0.1 1.0 6 0.4 10.6 6 1.3pqqCTTn3-gusA306 1.3 6 0.3 43.1 6 2.5 0.4 6 0.1 8.5 6 0.1 1.6 6 0.8 21.3 6 1.1pqqDTTn3-gusA433 1.9 6 0.5 27.1 6 1.2 1.2 6 0.4 0.2 6 0.1 1.0 6 0.2 31.4 6 1.4pqqETTn3-gusA109 1.0 6 0.2 20.4 6 1.1 0.6 6 0.2 0.2 6 0.1 0.9 6 0.1 18.0 6 1.0pqqFTTn3-gusA96 1.2 6 0.7 35.9 6 2.3 0.9 6 0.3 0.2 6 0.1 2.7 6 0.7 21.3 6 2.1pqqHTTn3-gusA3 1.3 6 0.3 10.8 6 0.9 – – – 2

pqqITTn3-gusA24 1.3 6 0.2 11.4 6 1.3 1.1 6 0.4 0.2 6 0.1 13.1 6 0.4 19.2 6 1.7pqqJTTn3-gusA316 5.1 6 1.2 90.0 6 2.1 2.8 6 0.6 38.8 6 2.4 28.5 6 1.4 115.6 6 4.3pqqMTTn3-gusA117 1.5 6 0.2 15.6 6 1.5 1.2 6 0.5 0.2 6 0.1 1.1 6 0.1 9.4 6 1.2None 1.1 6 0.2 0.2 6 0.1 1.2 6 0.2 0.3 6 0.1 1.3 6 0.2 0.2 6 0.1





(Fig. 5B). Electrospray ionization (ESI)-mass spectrom-etry (MS) analysis of the peak fraction corresponding to5-acetonyl-PQQ from B16 culture filtrates and a stan-dard revealed [M-H]2 ions at mass-to-charge ratio 387(Fig. 5C). This result confirmed that wild-type B16produces PQQ in vitro. None of the mutants defectivein plant growth promotion produced PQQ, andpOK53, which carries all of the pqq genes, conferredPQQ production in the mutants (one example is shownin Fig. 5B).

To confirm that PQQ promotes plant growth, syn-thetic PQQ was applied to germinating seedlings ofthe cucumber cultivar ‘Eunsungbagdadagi’, becausetomato seedlings did not grow well on Murashige andSkoog medium or in sand. Concentrations of syntheticPQQ ranging from 0 to 1,000 nM were used. Significantincreases in the fresh weight were observed in cucum-ber seedlings growing on Murashige and Skoogmedium that were treated with PQQ concentrations.100 nM (Fig. 6A). Cucumber seedlings growing in sandtreated with synthetic PQQ showed significantly greaterfresh weight than seedlings growing in Murashige andSkoog medium (Fig. 6B). Treatment with 50, 100, or1,000 nM PQQ increased the fresh weight of cucumberseedlings growing in sand by 18.4%, 17.1%, and 23.9%,respectively (Fig. 6). To determine whether PQQ pro-motes the growth of various plants, synthetic PQQ was

applied to germinating seedlings of Arabidopsis eco-type Columbia and the hot pepper cultivar ‘Bukang’on Murashige and Skoog medium. The fresh and dryweights of Arabidopsis and the size of the cotyledonsof hot pepper treated with 25 nM PQQ were increased(data not shown).

PQQ Scavenges ROS and H2O2 in Cucumber Leaves

To determine possible biochemical mechanisms in-volved in the promotion of plant growth by PQQ, weevaluated the ability of wild-type B16 and PQQ inplanta to scavenge ROS and hydrogen peroxide (H2O2)using nitroblue tetrazolium (NBT) and diaminobenzi-dine (DAB) staining, respectively. Wounded leaf discsof the cucumber cultivar ‘Eunsungbagdadagi’ treatedwith the PQQ-deficient mutant strain BK433 or waterclearly showed higher ROS production than leaf discsfrom plants treated with wild-type B16 (Fig. 7A). Thedeposition of blue formazan, an indication of ROSproduction in leaf discs, decreased as the PQQ con-centration exceeded 100 nM (Fig. 7A). Wounded leafdiscs were stained with DAB to locate H2O2, and lessH2O2 accumulation was observed in leaf discs treatedwith wild-type B16 than with the PQQ-deficient mu-tant BK433 or water (Fig. 7B). Staining was much lessintense after treatment with 100 or 1,000 nM synthetic

Figure 4. Effect of wild-type P.fluorescens B16 and mutant K818on the growth and yield of tomatoin hydroponic culture in 2002. A,Height. B, Number of flowers. C,Accumulated fruit numbers ofseven harvests. D, Total weight offruits per harvest. Vertical bars in-dicate SD. Data are the average ofthree replications (three plants perreplication) for each treatment. Dif-ferent letters indicate significantdifferences between the treatmentsaccording to Fisher’s protected LSD

test (P 5 0.05).

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PQQ than after water treatment, suggesting that PQQeffectively scavenged H2O2 in wounded cucumber leaves.

To determine whether PQQ affects the activity ofantioxidant enzymes, such as superoxide dismutase(SOD), ascorbate peroxidase (APX), and catalase, cu-cumber leaf extracts treated with synthetic PQQ, wild-type strain B16, or the PQQ-deficient mutant strainBK433 were examined using active staining methods

in native PAGE. There were no detectable differencesin the SOD, APX, or catalase activities among the treat-ments (data not shown).

Mineral Phosphate Solubilization Activity

To determine the mineral phosphate solubilization(MPS) activities of wild-type B16 and pqqTTn3-gusA

Figure 5. Analysis of PQQ synthesized by wild-type strain P. fluorescens B16 and the PQQ-deficient mutant BK433. A, Structureof PQQ and 5-acetonyl-PQQ (PQQ derivatized with acetone). B, HPLC detection of PQQ and 5-acetonyl-PQQ. Arrows indi-cate 5-acetonyl-PQQ. C, Negative-mode ESI-MS of 5-acetonyl-PQQ from synthetic PQQ and purified PQQ from wild-typestrain B16.





mutants, we performed MPS assays on Glc minimalmedium agar plates containing tricalcium phosphate(TCP). Both wild-type B16 and all pqqTTn3-gusA mu-tants showed very low MPS activity, with no signifi-cant differences (data not shown).

DISCUSSION

Plant growth promotion by PGPR has receivedattention for academic and practical reasons becausebeneficial interactions between PGPR and plants offertremendous potential for field applications. To be aneffective PGPR, an organism must be able to colonizeroots because the organism needs to establish itself inthe rhizosphere at population densities sufficient toproduce a beneficial effect. Thus, previous failures inplant growth promotion studies in the field have oftenbeen correlated with poor root colonization (Bloembergand Lutenberg, 2001). We found that the plant growthpromotion-deficient mutant K818 maintained the

ability to colonize roots, which suggested that addi-tional factors beyond root colonization are required forplant growth promotion. This result led us to identifya new plant growth promotion factor, PQQ, fromP. fluorescens B16.

In this study, we identified four previously uniden-tified pqq genes. It is unclear how these genes areinvolved in the biochemical pathways of PQQ biosyn-thesis. Possible PQQ biosynthesis pathways startingwith a Tyr and a Glu residue have been proposedbecause the small PqqA peptide contains a Glu and aTyr residue at conserved positions (Houck et al., 1991).If this is the case, PqqA might be a precursor in PQQbiosynthesis, which would require its synthesis instoichiometric amounts rather than catalytic amountsof other Pqq proteins involved in the biosynthesis ofPQQ. This is supported by the fact that expression ofpqqA is higher than that of the other pqq genes. Ex-pression of pqq genes depended on PqqH, which ishighly similar to a LysR-type regulator, but from thisstudy we do not know whether PqqH requires a

Figure 6. Growth promotion of cucumber treatedwith synthetic PQQ. Cucumber plants grown inMurashige and Skoog medium (A) or sand (B) con-taining 5, 50, 100, or 1,000 nM PQQ are shown. C,Fresh weight of cucumber treated with syntheticPQQ in experiments A and B above. Photographswere taken 13 d after transplanting. All values aremeans from triplicate experiments. Values in the plotfollowed by the same letter are not significantlydifferent according to Fisher’s protected LSD test(P 5 0.05). Bar 5 5 cm.

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coinducer, as do other LysR-type regulators. PqqK ispredicted to be a DNA-binding protein, but its bio-chemical role remains to be clarified.

The fact that the expression of pqq genes is regulatedby PqqH under nutrient-limiting conditions is consis-tent with other reports that PGPR are often effectiveunder low-nutrient conditions and have little or nomeasurable effect on plant growth when the plants aregrown in nutrient-rich soil under optimal conditions(Penrose and Glick, 2003). This result also explainswhy both wild-type B16 and PQQ promote plantgrowth effectively on rockwool, a hydroponic culturesystem, and in sand. Our findings suggest that thenutritional level of soil is critical for screening PGPRcandidates.

Some information on the biochemical functions ofPQQ has been reported. It is known that PQQ is acofactor of aminoadipic 6-semialdehyde-dehydrogenase(U26), which is involved in Lys degradation in mice.Numerous studies have reported that PGPR is able to

solubilize inorganic and/or organic phosphates in soilfollowing formation of the GDH-PQQ holoenzyme(Liu et al., 1992). However, the fact that wild-type B16and pqqTTn3-gusA mutants exhibited very low andapproximately the same MPS activity suggests thatPQQ plays a role beyond that of acting as a cofactor ofthe PQQ-dependent dehydrogenase for plant growthpromotion.

In addition, PQQ acts as an antioxidant in animalcells, preventing cell injury (Smidt et al., 1991). E. colicells synthesizing PQQ showed increased expressionof antioxidant enzymes, such as catalase and SOD,conferring a high level of protection of cells againstphotodynamically produced ROS (Khairnar et al.,2003). There has also been a report that PQQ acts asa ROS scavenger by directly neutralizing reactivespecies, thereby protecting bacterial cells from oxida-tive stress (Misra et al., 2004). The direct scavenging ofROS by PQQ in cucumber leaves, as opposed to PQQenhancing the activities of antioxidant enzymes, as

Figure 7. Microscopic detection ofROS (A) and H2O2 (B) in cucumberleaf discs. Leaf discs were treatedwith water (a), wild-type B16 (b),PQQ-deficient mutant strain BK433(c), 10 nM PQQ (d), 100 nM PQQ (e),or 1,000 nM PQQ (f). Insets showwhole leaf discs stained with NBT(A) or DAB (B). Third leaves of cu-cumber seedlings were stained withNBTor DAB at 7 d after inoculationwith bacteria. Eight leaf discs wereused for each treatment. Blue colorindicates the formation of insolubleformazan deposits that are pro-duced when NBT reacts with ROS.The deep-brown color is producedby the reaction of DAB with H2O2.The experiment was repeated threetimes with consistent results. Bar 5

200 mm.





observed in bacteria and rats, could be strengthenedby molecular biological means that might support ourleaf-disc assay data obtained through staining. How-ever, no molecular gene markers related to cucumberantioxidant enzyme genes were available for use innorthern-blot analysis. Nonetheless, our results areconsistent with PQQ directly scavenging superoxide(Urakami et al., 1997). In addition, PQQ may serve as adirect electron acceptor in reactions with reactivenitrogen species, thus protecting neurons against thetoxicity of peroxynitrite in rat forebrain neurons dur-ing culture (Zhang and Rosenberg, 2002).

PQQ is at least 100 times more efficient than ascorbicacid, isoflavonoids, and polyphenolic compounds inassays assessing redox cycling potentials (Stites et al.,2000). In addition to scavenging superoxide, PQQ couldalso scavenge other toxic free radicals, as do vitamin E,b-carotene and carotenoids, vitamin C, flavonoids,conjugated linoleic acid, and phenolic compounds(McIntire, 1998). Therefore, the antioxidant PQQ prob-ably confers plant growth promotion at the very lowconcentrations produced by the wild-type strain B16.

There have been few studies of the functional rolesof PQQ in plants. It is known that PQQ stimulatespollen germination in vitro in the plant species Lilium,Tulipa, and Camellia (Xiong et al., 1988, 1990), but themechanisms are unclear. This study provides evidencethat PQQ is a plant growth-promotion factor becauseof its antioxidant activity. Therefore, we believe thatthe biochemical basis of plant growth promotionmediated by PQQ is similar to that of its growthpromotion in mammals. It would be worthwhile toinvestigate the wide range of PGPR for PQQ produc-tion. We expect that many PGPR produce PQQ, whichwould illuminate previously unknown plant growth-promotion mechanisms. As in mammals, PQQ hasgreat potential to be used as a growth-promotionfactor in plants.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions

Bacterial strains and plasmids used in this study are listed in Table I.

Escherichia coli strains were cultured on LB medium at 37�C. Pseudomonas

fluorescens strain B16 was routinely cultivated at 28�C on LB medium or AB

minimal medium (0.3% K2HPO4, 0.1% NaH2PO4, 0.1% NH4Cl, 0.03%

MgSO4�7H2O, 0.015% KCl, 0.01% CaCl2�2H2O, 0.00025% FeSO4�7H2O, pH

7.0) supplemented with 0.2% Glc. Antibiotics were used at the following

concentrations: ampicillin, 100 mg mL21; chloramphenicol, 34 mg mL21;

gentamycin, 50 mg mL21; kanamycin, 50 mg mL21; nalidixic acid, 20 mg

mL21; rifampicin, 50 mg mL21; spectinomycin, 50 mg mL21; and tetracycline,

50 mg mL21.

DNA Manipulation and Transposon Mutagenesis

Standard methods were used for DNA cloning, restriction mapping, and

gel electrophoresis as described by Sambrook et al. (1989). The suicide plasmid

pJFF350 (Fellay et al., 1989) was used to generate transposon insertions in the

chromosome of strain B16. Because the VKm element carries an origin of

replication and no EcoRI site, 1 mg of the total genomic DNA of the mutants

was digested with EcoRI, self ligated, and transformed into E. coli DH5a,

followed by selection on LB agar medium containing kanamycin, to rescue the

region flanking the insertion. The flanking region was sequenced with the

primer HR (5#-TGCTTCAATCAATCACCGG-3#). pOK40 and pOK53, which

carry all of the PQQ biosynthetic genes, were mutagenized with Tn3-gusA as

described by Bonas et al. (1989). The insertion site and orientation of Tn3-gusA

in each mutant were mapped by restriction enzyme digestion analysis and

direct sequencing of the plasmid using the primer Tn3gus (5#-CCGGTCATCT-

GAGACCATTAAAAGA-3#), which allows sequencing from the Tn3-gusA

sequence. To generate a pqqH mutant, the V fragment was inserted into the

PshAI site of pOK40, which carries the pqqH gene, followed by cloning into

pRK415, resulting in pOK67. Mutagenized plasmids carrying Tn3-gusA in-

sertions or the V fragment were introduced individually into the parent strain

B16 by conjugation and marker exchanged into wild-type strain B16 as

described (Fellay et al., 1989). All marker exchanges were confirmed by

Southern hybridization analysis.

DNA Sequencing and Data Analysis

The 13.4-kb insert in pOK51, carrying all of the pqq genes, was digested

with appropriate restriction enzymes and subcloned into the corresponding

sites in pBluescriptII SK1. DNA fragments were sequenced using the BigDye

terminator kit (Applied Biosystems) with the universal and reverse primers.

Synthetic primers were designed for primer walking when necessary. DNA

sequences were assembled and ORFs were identified using the SeqManII

subroutine of DNASTAR. All potential ORFs larger than 249-bp were exam-

ined for possible ribosome-binding sites and annotated using the BLASTX and

BLASTP protocols (Altschul et al., 1990). DNA sequences were analyzed using

the BLAST program at the National Center for Biotechnology Information

(Gish and States, 1993), MEGALIGN (DNASTAR), and GENETYX-WIN

(Software Development, Inc.).

GUS Assay

The GUS enzyme assay (Jefferson et al., 1987) was performed with some

modifications. All strains of P. fluorescens B16 were grown in AB minimal

medium containing 0.04% gluconic acid, centrifuged, resuspended in GUS

extraction buffer, and lysed by sonication with a VCX-400 sonicator (Sonics

and Materials, Inc.). The extract was subjected to GUS enzyme assay with

4-methylumbelliferyl glucuronide as the substrate. Fluorescence was measured

at 365 nm for excitation and 460 nm for emission in a TKO100 fluorometer

(Hoefer Scientific Instruments). One unit of GUS was defined as 1 nmol of

4-methylumbelliferone released per bacterium per minute.

Plant Growth Promotion and RootColonization Measurements

Tomato (Solanum lycopersicum ‘Kwangsoo’) seeds were grown to the four-

or five-leaf stage in rockwool plugs. Root systems of the seedlings were

immersed for 1 h in bacterial suspensions (108 CFU mL21) for bacterization

and then transplanted into rockwool cubes (10 3 10 3 7 cm) and kept in a

greenhouse at 25�C 6 3�C. One-half-strength hydroponic culture solution

(COSEAL) was supplied twice per week. Rockwool cubes were arranged in a

randomized design. Replicated field trials were conducted over 2 years in the

hydroponic culture system (12.6 3 1.9 m). Trials were carried out under

natural illumination at 25�C 6 3�C from December, 2002 to April, 2003 for the

first year and from September, 2003 to January, 2004 for the second year.

Tomato seedlings at the four- or five-leaf stage in rockwool plugs were treated

with bacterial suspensions (108 CFU mL21) and transferred into the hydro-

ponic culture system. The bacterial suspension (108 CFU mL21) was then

applied to the plants seven times at 10-d intervals after transplanting to

provide sufficient bacterial cells and to ensure that the size of the bacterial

population was not a limiting factor. One-half-strength hydroponic culture

solution was supplied five times per day for 2 min. Tomato plants were grown

for 5 months.

Plant growth promotion was evaluated under two different conditions. In

rockwool cubes, the height of the tomato plants was measured 21 d after in-

oculation. Root samples collected from rockwool cubes at 21 d were macerated

in a sterile mortar and pestle. The population density of the bacteria on the

roots was determined by dilution plate counting. In the hydroponic culture

system, the height, thickness, number of stems, and number of flowers were

recorded at 7-d intervals and mature tomato fruits were harvested seven

times.

Choi et al.




PQQ Analysis

To measure PQQ production, bacteria were grown for 48 h at 28�C in AB

minimal medium containing 0.4% gluconic acid. One volume of cell culture

was diluted with nine volumes of methanol and the precipitated materials

were removed by centrifugation. After evaporation of the methanol, a Sep-Pak

C18 cartridge (Waters) was washed with 10 mL of methanol and subsequently

with 10 mL of water. The sample was acidified with HCl to pH 2.0 and loaded

onto the cartridge. After washing with 10 mL of 2 mM HCl, PQQ was eluted

with 70% methanol. To identify the peak of PQQ, 200 mL of the sample were

mixed with 100 mL of 0.2 M Na2B4O7 buffer and adjusted to pH 8.0 with HCl

and 90 mL of 0.5% (v/v) acetone. RP-HPLC was performed using a Shimadzu

LC-6A HPLC system as described previously (Van der Meer et al., 1990) with a

fluorescence detector. Fluorescence was monitored at ex 5 360 and em 5 480

nm. A C18 column (150 mm 3 4.6 mm i.d., 5-mm particle size; Phenomenex)

was used for analytical separation. Fractions corresponding to the acetone

adduct (5-acetonyl-PQQ) were analyzed using ESI-MS (JEOL).

Plant Growth Promotion by Synthetic PQQ

Arabidopsis (Arabidopsis thaliana ecotype Columbia), hot pepper (Capsicum

annum ‘Bukang’), and cucumber (Cucumis sativus ‘Eunsungbagdadagi’) seeds

were surface sterilized (70% ethanol for 5 min followed by 1% sodium

hypochlorite for 15 min), rinsed 10 times with sterile, distilled water, placed on

petri dishes containing medium consisting of one-half-strength Murashige

and Skoog salts (Sigma), 0.4% agar, and 3% Suc, and allowed to germinate

over 2 d at 28�C. Glass bottles (8.5 3 16 cm) were prepared with one-half-

strength Murashige and Skoog medium containing 5, 50, 100, or 1,000 nM of

synthetic PQQ (Sigma). Two-day-old cucumber seedlings were transferred

into the glass bottles. Germinated Arabidopsis and hot pepper seedlings were

transferred to the glass bottles with one-half-strength Murashige and Skoog

medium containing 25 nM of synthetic PQQ. Water was used as a control.

Glass bottles were arranged in a randomized design.

Sand was rinsed in distilled water for 3 d and autoclaved twice. Cucumber

seeds were surface sterilized and placed in petri dishes containing sterile

water to germinate at 28�C. Two-day-old cucumber seedlings were immersed

for 1 h in 10 mL of 5, 50, 100, or 1,000 nM synthetic PQQ, transferred into the

sand, and the surplus synthetic PQQ solution that remained after treatment

was poured into the sand. Water was used as a control. Glass bottles and

plants transplanted in sand were placed in a growth chamber set to a 14-h-

light/10-h-dark cycle at 24�C 6 1�C with a relative humidity of 60%. Fresh

weight of the plants was recorded at 13 d after transplanting.

Detection of Localized Accumulation of ROS and H2O2

in Leaf Discs

Cucumbers were grown until the three-leaf stage in rockwool plugs. Root

systems of the seedlings were immersed for 1 h in a bacterial suspension (108

CFU mL21) for bacterization. Eight leaf discs (7 mm in diameter) from the

third leaves of cucumber seedlings were used for detection of ROS and H2O2

7 d after inoculation. For PQQ treatment, leaf discs were immersed for 14 h at

25�C in 0, 10, 100, or 1,000 nM synthetic PQQ. All leaf discs were vacuum

infiltrated with 1 mg mL21 NBT in 10 mM potassium phosphate buffer (pH 7.8)

or DAB solution and incubated at 25�C under light for 2 h. Leaf discs were

rinsed with 80% (v/v) ethanol for 10 min at 70�C, mounted on a glass slide in

lactic acid:phenol:water (1:1:1 [v/v/v]), and photographed directly using a

microscope (Carl Zeiss).

Native-PAGE Analysis of SOD, APX, andCatalase Activities

For determination of antioxidant enzyme activities, cucumber leaves (1 g)

were frozen in liquid nitrogen, ground, and resuspended in 150 mL of 50 mM

KH2PO4 (pH 7.8). The homogenate was centrifuged at 13,000g for 15 min and

protein content of the supernatant was determined (Bradford protein assay;

Bio-Rad). Samples of 30 mg of protein from each tissue homogenate were

separated in 10% native polyacrylamide gels. SOD activity in the gels was

determined using the modified staining method (McCord and Fridovich,

1969). Gels were held in darkness for 30 min in a 1:1 mixture of 0.06 mM

riboflavin 1 0.651% (w/v) TEMED and 2.5 mM NBT, both in 50 mM phosphate

buffer at pH 7.8, and then developed for 20 min under moderate light

conditions. APX and catalase activities were detected using previously

described procedures (Wayne and Diaz, 1986; Mittler and Zilinskas, 1993).

Mineral Phosphate Solubilizing Activity Assay

MPS activity of bacteria was checked on Glc minimal medium agar plates

containing TCP, as described previously (Krishnaraj and Goldstein, 2001).

Bacterial cultures were grown overnight and approximately 5 3 108 CFU

mL21 cells of each bacterium were spotted on a TCP agar plate. Plates were

incubated at 28�C for 48 h. Formation of clearing halos in the plates was

recorded.

Statistical Analysis

Experimental data were analyzed statistically using ANOVA (SAS Insti-

tute). Significance of the effect of treatment was determined by the magnitude

of the F value (P 5 0.05). When a significant F test was obtained for the

treatments, separation of means was accomplished by Fisher’s protected LSD.

Sequence data from this article can be found in the GenBank data libraries

under accession number AY780887.

Received November 6, 2007; accepted November 20, 2007; published Novem-

ber 30, 2007.

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