Pyrroloquinoline Quinone Is a Plant Growth ... | Plant Physiology

Pyrroloquinoline Quinone Is a Plant Growth Promotion Factor 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 National University, 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, Korea Research 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 growth promotion by this organism, we mutagenized wild-type strain B16 using VKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqHdependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces 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, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 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 B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.

Bacteria that colonize plant roots and enhance plant growth by any mechanism are referred to as plant growth-promoting rhizobacteria (PGPR). PGPR have been applied on various crops to enhance growth, seed emergence, crop yield, and disease control, and some have been commercialized (Kloepper, 1992; Glick, 1995; Dey et al., 2004). The use of PGPR in sustainable agriculture is steadily increasing and offers an attractive way to replace chemical fertilizers, pesticides, and supplements. PGPR can promote plant growth indirectly or directly. Indirect plant growth promotion is mediated by antibiotics or siderophores produced by PGPR that decrease or prevent the deleterious effects of plantpathogenic microorganisms (Leong, 1986; Sivan and 1 This work was supported by the Crop Functional Genomics Center of the 21st Century Frontier R&D Program (grant no. CG2131), funded by the Ministry of Science and Technology of the Republic of Korea, and by a Korea Research Foundation Grant, funded by the Korean Government (Ministry of Education and Human Resources Development, Basic Research Promotion Fund; grant no. KRF–2006–005–J04701). * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ingyu Hwang ([email protected]). www.plantphysiol.org/cgi/doi/10.1104/pp.107.112748

Chet, 1992). Direct plant growth-promoting factors include various phytohormones (Xie et al., 1996), solubilization of soil phosphorus and iron (De Freitas et al., 1997), N2 fixation (Christiansen-Weneger, 1992), increases in nitrate uptake (Sophie et al., 2006), reduction of membrane potential in roots (Bashan and Levanony, 1991), 1-aminocyclopropane-1-carboxylate deaminase (which modulates plant growth and development; Safronova et al., 2006), and the production of volatiles as potential signal molecules (Ryu et al., 2003). In mammals, pyrroloquinoline quinone (PQQ) functions as a potent growth factor, although its biological functions are not fully understood (Smidt et al., 1991; Steinberg et al., 1994). PQQ has attracted considerable interest because of its presence in a wide variety of foods and its remarkable antioxidant properties (Smidt et al., 1991; Kumazawa et al., 1995; Mitchell et al., 1999; He et al., 2003). PQQ is found in plant and animal tissues in the nanogram-to-gram range even though plants 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 as a reactive oxygen species (ROS) scavenger by directly neutralizing reactive species in Escherichia coli (Misra et al., 2004). PQQ acts as a noncovalently bound redox cofactor of several bacterial dehydrogenases, including methanol dehydrogenase and Glc dehydrogenase

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(GDH; Duine et al., 1990; Stites et al., 2000). GDH is a quinoprotein that uses PQQ as a cofactor and is involved in the periplasmic oxidation of Glc to gluconic acid, resulting in the solubilization of the poorly soluble calcium phosphate (Babu-Khan et al., 1995). In mammals, picomolar amounts of PQQ enhance DNA synthesis activity in human fibroblasts and display nerve growth factor-inducing activity (Naito et al., 1993; Yamaguchi et al., 1993). PQQ-deficient diets impair growth, cause immunological defects, and decrease fertility in mice (Killgore et al., 1989; Steinberg et al., 1994). Recently, PQQ has been proposed to function as a vitamin in mammals, following the identification of the first potential eukaryotic PQQ-dependent enzyme (Kasahara and Kato, 2003). However, this idea is still controversial and it is unknown whether PQQ affects plant development and growth in vivo. The biochemical pathways of PQQ biosynthesis are not fully understood, but it is known that Glu and Tyr are precursors (Houck et al., 1991; Unkefer et al., 1995). Genes involved in PQQ biosynthesis have been identified from various bacteria, including Acinetobacter calcoaceticus (Goosen et al., 1989), Methylobacterium extorquens AM1 (Toyama et al., 1997), Klebsiella pneumoniae (Meulenberg et al., 1992), Gluconobacter oxydans (Felder et al., 2000), and Pseudomonas fluorescens CHA0 (Schnider et al., 1991). The pqqABCDEF genes are conserved in bacteria, but the biochemical functions of the encoded proteins are largely unclear. Recently, PqqC has been reported to be the final catalyst in the production of PQQ (Magnusson et al., 2004). We have studied the promotion of plant growth by P. fluorescens B16, which was isolated from the roots of graminaceous plants. The wild-type B16 colonizes the roots of various plants and produces an antibacterial compound that is effective against plant root pathogens, such as Agrobacterium tumefaciens and Ralstonia solanacearum (Kang and Park, 1997; Kim et al., 1998; Kim et al., 2003). This organism also significantly promotes the growth of cucumber (Cucumis sativus) and barley (Hordeum vulgare) under greenhouse and field conditions (Kim et al., 1998). However, the mechanism of plant growth promotion by this strain is unknown. In this study, we report that PQQ synthesized by P. fluorescens B16 is a key factor involved in growth promotion in tomato (Solanum lycopersicum), cucumber, Arabidopsis (Arabidopsis thaliana), and hot pepper (Capsicum annuum). Moreover, we report four previously unidentified pqq genes and demonstrate that expression of the pqq genes is regulated by a transcriptional activator, PqqH. This article reports that PQQ promotes plant growth in vivo. RESULTS Isolation of a Plant Growth Promotion-Defective Mutant

Following random mutagenesis of P. fluorescens B16 with VKm, the mutant K818 was isolated from 2,000 prototrophic colonies due to its failure to promote the 658

growth of the tomato cultivar ‘Kwangsoo’ in a rockwool system. Heights of the tomato plants were measured every 3 d up to 27 d after the treatment of tomato plants at the four- or five-leaf stage with K818 or wildtype B16. Mutant K818 failed to promote tomato growth, whereas the height of plants treated with B16 was increased by approximately 25% at 27 d after treatment (Fig. 1). To determine whether K818 was able to colonize tomato roots, bacterial populations on the roots were examined. The colonizing populations of B16 and the mutant strain K818 on roots were 5.2 3 105 and 5.3 3 105 colony-forming units (CFU) g21 roots, respectively, indicating that the plant growth promotion-defective mutant K818 has the same rootcolonizing activity as wild-type B16. Identification of Genes Responsible for Plant Growth Promotion

To identify genes that confer plant growth promotion in wild-type B16, the DNA region flanking the VKm insertion in the mutant K818 was isolated by self ligation of chromosomal DNA digested with EcoRI. The rescued plasmid pOK8 had an insert of 5.8 kb and a 2.8-kb PstI fragment from pOK8 was subcloned into pBluescriptII SK1, resulting in pOK12. Analysis of the DNA sequences of the flanking regions from pOK12 revealed that the VKm element in the mutant K818 was inserted in a gene homologous to a LysR-type transcriptional regulator of P. fluorescens Pf0-1 (Tables I and II; Fig. 2). The cosmid clone pOK40, spanning the flanking regions of the gene disrupted in K818, was isolated from a genomic library of wild-type B16 by

Figure 1. Tomato plant growth promotion following treatment with wild-type P. fluorescens B16, the mutant K818, and K818 carrying pOK40. The height of the tomato plants was recorded at 3-d intervals up to 31 d after inoculation. The values are means of three replications per experiment pooled from three experiments. Vertical bars indicate SDs. Plant Physiol. Vol. 146, 2008

Pyrroloquinoline Quinone as a Plant Growth Promotion Factor

Table I. Bacterial strains and plasmids Ampr, Ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance; Nalr, nalidixic acid resistance; Rif r, rifampicin resistance; Smr, streptomycin resistance; Spr, spectinomycin resistance; Tetr, tetracycline resistance. Strain or Plasmid

E. coli DH5a S17-1 C2110 HB101 P. fluorescens B16 K818 BK1 BK544/BKS544 BK4/BKS4 BK306/BKS306 BK433/BKS433 BK109/BKS109 BK96/BKS96 BK24/BKS24 BK316/BKS316 BK175/BKS175 BK117/BKS117 Plasmids pBluescriptII SK1 pLAFR3 pRK415 pHoKmGus pSShe pHP45V pOK8 pOK12 pOK40 pOK51 pOK53 pOK59 pOK67

Characteristics

F2 F80 dlacZDM15D(lacZYA-argF)U169 endA1 recA1 hsdR17 (rk2mk1) deoR thi-1 supE44l2gyrA96 relA1 Tra1, recA, Spr polA, Nalr F- mcrB mrr hsdS20(rB2mB2) recA13 leuB6 ara-14 proAe lacY1 galK2 xyl-5 mtl-1 rpsL20(Smr) supE44l2

Gibco BRL Simon et al. (1983) Stachel et al. (1985) Gibco BRL

Wild-type, Rif r B16TVKm B16 pqqHTV B16 pqqATTn3-gusA544/BK1 pqqATTn3-gusA544 B16 pqqBTTn3-gusA4/BK1 pqqBTTn3-gusA4 B16 pqqCTTn3-gusA306/BK1 pqqCTTn3-gusA306 B16 pqqDTTn3-gusA433/BK1 pqqDTTn3-gusA433 B16 pqqETTn3-gusA109/BK1 pqqETTn3-gusA109 B16 pqqFTTn3-gusA96/BK1 pqqFTTn3-gusA96 B16 pqqITTn3-gusA24/BK1 pqqITTn3-gusA24 B16 pqqJTTn3-gusA316/BK1 pqqJTTn3-gusA316 B16 pqqKTTn3-gusA175/BK1 pqqKTTn3-gusA175 B16 pqqMTTn3-gusA117/BK1 pqqMTTn3-gusA117

Kim This This This This This This This This This This This This

Cloning vehicle; phagemid, pUC derivative, Ampr Tra2, Mob1, RK2 replicon, Tetr Mob1, lacZ, Tetr Promoterless GUS gene, Kmr, Ampr Cmr V cassette, Spr, Smr 5.8-kb self-ligated EcoRI clone from K818 2.8-kb PstI fragment from pOK8 cloned into pBluescriptII SK1 27.4-kb DNA fragment from strain B16 cloned into pLAFR3 13.4-kb BamHI fragment from pOK40 cloned into pBluescriptII SK1 13.4-kb BamHI fragment from pOK40 cloned into pLAFR3 2.0-kb BamHI-HindIII fragment including the pqqH region from pOK58 cloned into pLAFR3 14-kb HindIII fragment harboring a 2.0-kb V cassette in the PshAI site within pqqH in pOK40 cloned into pRK415

Stratagene Staskawicz et al. (1987) Keen et al. (1988) Bonas et al. (1989) Stachel et al. (1985) Prentki and Krisch (1984) This study This study This study This study This study This study

colony hybridization using the 1.8-kb HindIII/PstI fragment of pOK12 as a probe (Fig. 2). When pOK40 was mobilized into the mutant K818, the plant growthpromoting effect was restored to that of wild-type B16 (Fig. 1). A restriction enzyme map of the 25-kb insert of pOK40 was constructed (Fig. 2). To further characterize the insert, we mutagenized pOK40 and pOK53, which carries a 13.4-kb BamHI fragment from pOK40 in pLAFR3, with Tn3-gusA, followed by marker exchange into wild-type B16. We identified 12 Tn3-gusA insertions that interfere with plant growth promotion, delineating the essential region for this function (Fig. 2). The 13.4-kb BamHI fragment from pOK40 was subcloned into pBluescriptII SK1, resulting in pOK51, for DNA sequencing. The sequence of the fragment contains 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 Plant Physiol. Vol. 146, 2008

Source or Reference

et al. (2003) study study study study study study study study study study study study

This study

production (Fig. 2). pqqA, B, C, D, E, F, and M genes exhibit strong similarity to those of the P. fluorescens strain Pf0-1 (Table II; Fig. 3). Proteins PqqH and I of P. fluorescens B16 are highly similar to a LysR-type transcriptional regulator and a class-III aminotransferase present upstream of the pqq gene cluster in P. fluorescens Pf0-1, respectively (Table II). pqqJ is predicted to encode a 13.6-kD protein exhibiting 53% identity and 68% similarity to a putative cytoplasmic protein from Salmonella enterica sp. enterica serovar Choleraesuis str. SC-B67 (Table II). PqqK is predicted to be a protein of 11.6 kD that is similar to a DNA-binding protein of Sinorhizobium meliloti 1021 (Table II). Expression of pqq Genes in a PqqH-Dependent Manner in Nutrient-Limiting Conditions

To determine how the pqq genes of P. fluorescens B16 are expressed, we analyzed their expression levels 659

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

No. of Amino Acid Residuesb

pqqA

24

pqqB

303

pqqC

250

pqqD

91

pqqE

425

pqqF

812

pqqH

306

pqqI

427

pqqJ

119

pqqK

106

pqqM

607

Putative Function [Organism] (GenBank Accession No.)

PQQ A biosynthesis protein [P. fluorescens] (CAA60731) PQQ biosynthesis protein PqqB [P. fluorescens Pf0-1] (YP_350886) PQQ biosynthesis protein PqqC [P. fluorescens Pf0-1] (YP_350887) PQQ biosynthesis protein PqqD [P. fluorescens Pf0-1] (YP_350888) PQQ biosynthesis protein PqqE [P. fluorescens Pf0-1] (YP_350889) Peptidase M16A, coenzyme PQQ biosynthesis protein PqqF [P. fluorescens Pf0-1] (YP_350884) Transcriptional regulator, LysR family [P. fluorescens Pf0-1] (YP_350893) Aminotransferase class-III [P. fluorescens Pf0-1] (YP_350892) Putative cytoplasmic protein [Salmonella enterica subsp. enterica serovar Choleraesuis str. SC-B67] (YP_218683) Probable DNA-binding protein [Sinorhizobium meliloti 1021] (NP_436416) Peptidases S9, prolyl oligopeptidase active site region [P. fluorescens Pf0-1] (YP_350890)

BLAST E Value

6e-06 4e-164 2e-143 1e-44 0.0 0.0 2e-153 0.0 1e-26

4e-31 0.0

a

b Unified nomenclature for genes that encode P. fluorescens B16 Pqq proteins. Putative function of an individual gene product is predicted based on homology to proteins of known function in other PQQ synthesis pathways.

using pqqTTn3-gusA fusion mutants grown in LuriaBertani (LB) medium or Agrobacterium minimal medium (AB). None of the pqq genes were expressed at high levels in LB medium, but each was expressed more strongly in AB medium (Table III). This result indicates that pqq genes are expressed only under nutrientlimiting conditions. Because PqqH shows similarity to

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

Figure 2. Organization of the pqqABCDEFHIJKM genes. White arrows indicate the positions and orientations of the PQQ biosynthesis genes. Vertical bars in the maps indicate the positions and orientations of the Tn3-gusA insertions, and the major phenotypes of the mutants are represented below the restriction map. The vertical bar with a black circle indicates the position of the 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. 660

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Figure 3. Comparison of the pqq gene clusters of P. fluorescens B16 with those from P. fluorescens Pf0-1, Klebsiella pneumoniae, 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 homologous encoded proteins. The organization and size of the genes are depicted based on nucleotide sequence data from GenBank. The following 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).

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

pOK40 did promote growth (Fig. 4). We repeated the growth study of tomato plants in the hydroponic culture system twice over 2 years and observed very similar results; therefore, only 1 year of data is presented in Figure 4. These results indicate that the growth promotion of tomato plants by wild-type B16 can be achieved in a hydroponic culture system.

Promotion of Tomato Plant Growth in a Hydroponic Culture System

Because wild-type B16 promoted the growth of tomato plants in a rockwool system, we tested whether it could promote tomato plant growth in a hydroponic culture system. In this system, wild-type B16 increased plant height by 19.8% and flower number by 42%, as measured at 65 d after treatment (Fig. 4). In addition, treatment with B16 increased the fruit number and total fruit weight after the final harvest by 41% and 36%, respectively (Fig. 4). As expected, the mutant K818 failed to confer growth promotion and K818 carrying

PQQ Is a Key Element for Plant Growth Promotion

Based on the fact that mutations in pqq genes abolished plant growth-promotion activity of wild-type B16, we examined whether the strain produces PQQ by analyzing culture supernatants using reverse-phase (RP)-HPLC. PQQ was detected as 5-acetonyl-PQQ by comparison with the elution times of synthetic PQQ and 5-acetonyl-PQQ from the RP-HPLC chromatograms

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. All values are means 6 SD of values from triplicate experiments. –, Not determined. Specific Activity of GUS (10211 CFU21) B16 (Wild Type)

Tn3-gusA Fusion LB

pqqATTn3-gusA544 pqqBTTn3-gusA4 pqqCTTn3-gusA306 pqqDTTn3-gusA433 pqqETTn3-gusA109 pqqFTTn3-gusA96 pqqHTTn3-gusA3 pqqITTn3-gusA24 pqqJTTn3-gusA316 pqqMTTn3-gusA117 None Plant Physiol. Vol. 146, 2008

12.7 1.6 1.3 1.9 1.0 1.2 1.3 1.3 5.1 1.5 1.1

6 6 6 6 6 6 6 6 6 6 6

BK1 (B16 pqqHTV) AB

1.2 0.6 0.3 0.5 0.2 0.7 0.3 0.2 1.2 0.2 0.2

396.7 25.9 43.1 27.1 20.4 35.9 10.8 11.4 90.0 15.6 0.2

6 6 6 6 6 6 6 6 6 6 6

LB

5.9 2.0 2.5 1.2 1.1 2.3 0.9 1.3 2.1 1.5 0.1

3.5 1.0 0.4 1.2 0.6 0.9 1.1 2.8 1.2 1.2

6 6 6 6 6 6 – 6 6 6 6

BK1 (B16 pqqHTV, pOK59)

AB

0.4 0.2 0.1 0.4 0.2 0.3 0.4 0.6 0.5 0.2

142.0 6 0.2 6 8.5 6 0.2 6 0.2 6 0.2 6 – 0.2 6 38.8 6 0.2 6 0.3 6

LB

3.4 0.1 0.1 0.1 0.1 0.1 0.1 2.4 0.1 0.1

25.5 1.0 1.6 1.0 0.9 2.7

6 6 6 6 6 6 – 13.1 6 28.5 6 1.1 6 1.3 6

AB

2.3 0.4 0.8 0.2 0.1 0.7 0.4 1.4 0.1 0.2

415.5 6 10.6 6 21.3 6 31.4 6 18.0 6 21.3 6 2 19.2 6 115.6 6 9.4 6 0.2 6

5.4 1.3 1.1 1.4 1.0 2.1 1.7 4.3 1.2 0.1 661

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Figure 4. Effect of wild-type P. fluorescens B16 and mutant K818 on the growth and yield of tomato in hydroponic culture in 2002. A, Height. B, Number of flowers. C, Accumulated fruit numbers of seven harvests. D, Total weight of fruits per harvest. Vertical bars indicate SD. Data are the average of three replications (three plants per replication) for each treatment. Different letters indicate significant differences between the treatments according to Fisher’s protected LSD test (P 5 0.05).

(Fig. 5B). Electrospray ionization (ESI)-mass spectrometry (MS) analysis of the peak fraction corresponding to 5-acetonyl-PQQ from B16 culture filtrates and a standard revealed [M-H]2 ions at mass-to-charge ratio 387 (Fig. 5C). This result confirmed that wild-type B16 produces PQQ in vitro. None of the mutants defective in plant growth promotion produced PQQ, and pOK53, which carries all of the pqq genes, conferred PQQ production in the mutants (one example is shown in Fig. 5B). To confirm that PQQ promotes plant growth, synthetic PQQ was applied to germinating seedlings of the cucumber cultivar ‘Eunsungbagdadagi’, because tomato seedlings did not grow well on Murashige and Skoog medium or in sand. Concentrations of synthetic PQQ ranging from 0 to 1,000 nM were used. Significant increases in the fresh weight were observed in cucumber seedlings growing on Murashige and Skoog medium that were treated with PQQ concentrations .100 nM (Fig. 6A). Cucumber seedlings growing in sand treated with synthetic PQQ showed significantly greater fresh weight than seedlings growing in Murashige and Skoog medium (Fig. 6B). Treatment with 50, 100, or 1,000 nM PQQ increased the fresh weight of cucumber seedlings growing in sand by 18.4%, 17.1%, and 23.9%, respectively (Fig. 6). To determine whether PQQ promotes the growth of various plants, synthetic PQQ was 662

applied to germinating seedlings of Arabidopsis ecotype Columbia and the hot pepper cultivar ‘Bukang’ on Murashige and Skoog medium. The fresh and dry weights of Arabidopsis and the size of the cotyledons of 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 involved in the promotion of plant growth by PQQ, we evaluated the ability of wild-type B16 and PQQ in planta to scavenge ROS and hydrogen peroxide (H2O2) using nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining, respectively. Wounded leaf discs of the cucumber cultivar ‘Eunsungbagdadagi’ treated with the PQQ-deficient mutant strain BK433 or water clearly showed higher ROS production than leaf discs from plants treated with wild-type B16 (Fig. 7A). The deposition of blue formazan, an indication of ROS production in leaf discs, decreased as the PQQ concentration exceeded 100 nM (Fig. 7A). Wounded leaf discs were stained with DAB to locate H2O2, and less H2O2 accumulation was observed in leaf discs treated with wild-type B16 than with the PQQ-deficient mutant BK433 or water (Fig. 7B). Staining was much less intense after treatment with 100 or 1,000 nM synthetic Plant Physiol. Vol. 146, 2008


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

PQQ than after water treatment, suggesting that PQQ effectively scavenged H2O2 in wounded cucumber leaves. To determine whether PQQ affects the activity of antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase, cucumber leaf extracts treated with synthetic PQQ, wildtype strain B16, or the PQQ-deficient mutant strain BK433 were examined using active staining methods Plant Physiol. Vol. 146, 2008

in native PAGE. There were no detectable differences in the SOD, APX, or catalase activities among the treatments (data not shown). Mineral Phosphate Solubilization Activity

To determine the mineral phosphate solubilization (MPS) activities of wild-type B16 and pqqTTn3-gusA 663

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Figure 6. Growth promotion of cucumber treated with synthetic PQQ. Cucumber plants grown in Murashige and Skoog medium (A) or sand (B) containing 5, 50, 100, or 1,000 nM PQQ are shown. C, Fresh weight of cucumber treated with synthetic PQQ in experiments A and B above. Photographs were taken 13 d after transplanting. All values are means from triplicate experiments. Values in the plot followed by the same letter are not significantly different according to Fisher’s protected LSD test (P 5 0.05). Bar 5 5 cm.

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

DISCUSSION

Plant growth promotion by PGPR has received attention for academic and practical reasons because beneficial interactions between PGPR and plants offer tremendous potential for field applications. To be an effective PGPR, an organism must be able to colonize roots because the organism needs to establish itself in the rhizosphere at population densities sufficient to produce a beneficial effect. Thus, previous failures in plant growth promotion studies in the field have often been correlated with poor root colonization (Bloemberg and Lutenberg, 2001). We found that the plant growth promotion-deficient mutant K818 maintained the 664

ability to colonize roots, which suggested that additional factors beyond root colonization are required for plant growth promotion. This result led us to identify a new plant growth promotion factor, PQQ, from P. fluorescens B16. In this study, we identified four previously unidentified pqq genes. It is unclear how these genes are involved in the biochemical pathways of PQQ biosynthesis. Possible PQQ biosynthesis pathways starting with a Tyr and a Glu residue have been proposed because the small PqqA peptide contains a Glu and a Tyr residue at conserved positions (Houck et al., 1991). If this is the case, PqqA might be a precursor in PQQ biosynthesis, which would require its synthesis in stoichiometric amounts rather than catalytic amounts of other Pqq proteins involved in the biosynthesis of PQQ. This is supported by the fact that expression of pqqA is higher than that of the other pqq genes. Expression of pqq genes depended on PqqH, which is highly similar to a LysR-type regulator, but from this study we do not know whether PqqH requires a Plant Physiol. Vol. 146, 2008


Figure 7. Microscopic detection of ROS (A) and H2O2 (B) in cucumber leaf discs. Leaf discs were treated with 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 show whole leaf discs stained with NBT (A) or DAB (B). Third leaves of cucumber seedlings were stained with NBT or DAB at 7 d after inoculation with bacteria. Eight leaf discs were used for each treatment. Blue color indicates the formation of insoluble formazan deposits that are produced when NBT reacts with ROS. The deep-brown color is produced by the reaction of DAB with H2O2. The experiment was repeated three times with consistent results. Bar 5 200 mm.

coinducer, as do other LysR-type regulators. PqqK is predicted to be a DNA-binding protein, but its biochemical role remains to be clarified. The fact that the expression of pqq genes is regulated by PqqH under nutrient-limiting conditions is consistent with other reports that PGPR are often effective under low-nutrient conditions and have little or no measurable effect on plant growth when the plants are grown in nutrient-rich soil under optimal conditions (Penrose and Glick, 2003). This result also explains why both wild-type B16 and PQQ promote plant growth effectively on rockwool, a hydroponic culture system, and in sand. Our findings suggest that the nutritional level of soil is critical for screening PGPR candidates. Some information on the biochemical functions of PQQ has been reported. It is known that PQQ is a cofactor of aminoadipic 6-semialdehyde-dehydrogenase (U26), which is involved in Lys degradation in mice. Numerous studies have reported that PGPR is able to Plant Physiol. Vol. 146, 2008

solubilize inorganic and/or organic phosphates in soil following formation of the GDH-PQQ holoenzyme (Liu et al., 1992). However, the fact that wild-type B16 and pqqTTn3-gusA mutants exhibited very low and approximately the same MPS activity suggests that PQQ plays a role beyond that of acting as a cofactor of the PQQ-dependent dehydrogenase for plant growth promotion. In addition, PQQ acts as an antioxidant in animal cells, preventing cell injury (Smidt et al., 1991). E. coli cells synthesizing PQQ showed increased expression of antioxidant enzymes, such as catalase and SOD, conferring a high level of protection of cells against photodynamically produced ROS (Khairnar et al., 2003). There has also been a report that PQQ acts as a ROS scavenger by directly neutralizing reactive species, thereby protecting bacterial cells from oxidative stress (Misra et al., 2004). The direct scavenging of ROS by PQQ in cucumber leaves, as opposed to PQQ enhancing the activities of antioxidant enzymes, as 665

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observed in bacteria and rats, could be strengthened by molecular biological means that might support our leaf-disc assay data obtained through staining. However, no molecular gene markers related to cucumber antioxidant enzyme genes were available for use in northern-blot analysis. Nonetheless, our results are consistent with PQQ directly scavenging superoxide (Urakami et al., 1997). In addition, PQQ may serve as a direct electron acceptor in reactions with reactive nitrogen species, thus protecting neurons against the toxicity of peroxynitrite in rat forebrain neurons during culture (Zhang and Rosenberg, 2002). PQQ is at least 100 times more efficient than ascorbic acid, isoflavonoids, and polyphenolic compounds in assays assessing redox cycling potentials (Stites et al., 2000). In addition to scavenging superoxide, PQQ could also 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 probably confers plant growth promotion at the very low concentrations produced by the wild-type strain B16. There have been few studies of the functional roles of PQQ in plants. It is known that PQQ stimulates pollen germination in vitro in the plant species Lilium, Tulipa, and Camellia (Xiong et al., 1988, 1990), but the mechanisms are unclear. This study provides evidence that PQQ is a plant growth-promotion factor because of its antioxidant activity. Therefore, we believe that the biochemical basis of plant growth promotion mediated by PQQ is similar to that of its growth promotion in mammals. It would be worthwhile to investigate the wide range of PGPR for PQQ production. We expect that many PGPR produce PQQ, which would illuminate previously unknown plant growthpromotion mechanisms. As in mammals, PQQ has great potential to be used as a growth-promotion factor 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% MgSO47H2O, 0.015% KCl, 0.01% CaCl22H2O, 0.00025% FeSO47H2O, 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

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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#-CCGGTCATCTGAGACCATTAAAAGA-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 insertions 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 examined 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 Root Colonization Measurements Tomato (Solanum lycopersicum ‘Kwangsoo’) seeds were grown to the fouror 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 hydroponic 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 inoculation. 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.



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-halfstrength 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-hlight/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, and Catalase 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 Institute). 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 November 30, 2007.

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