Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16

Abstract

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 OmegaKm 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 PqqH-dependent 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.

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.

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 ΩKm insertion in mutant strain K818. The vertical bar with a black triangle indicates the position of the Ω cassette insertion. B, BamHI; E, EcoRI; H, HindIII.

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).

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 = 0.05).

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.

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 = 0.05). Bar = 5 cm.

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 = 200 μm.