WO2006075807A1

WO2006075807A1 - Pyrroloquinoline quinone and its biosynthetic genes for plant growth promotion and uses thereof

Info

Publication number: WO2006075807A1
Application number: PCT/KR2005/000084
Authority: WO
Inventors: Ingyu Hwang; Chang-Seuk Park; Okhee Choi; Jinwoo Kim
Original assignee: Seoul National University Industry Foundation
Priority date: 2005-01-11
Filing date: 2005-01-11
Publication date: 2006-07-20

Abstract

The present invention relates to pyrroloquinoline quinone (PQQ) and its biosynthetic genes for plant growth and uses thereof. The biosynthetic genes of PQQ are derived from Pseudomonas fluorescens B16 and delivered for promoting plant growth.

Description

PYRROLOQUINOLINE QUINONE AND ITS BIOSYNTHETIC GENES FOR PLANT GROWTH PROMOTION AND USES THEREOF

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to plant growth- promotion factor derived from Pseudomonas fluorescens and uses thereof, particularly to DNA molecules promoting plant growth and a method of promoting plant growth by applying the plant growth-promotion factor .

DESCRIPTION OF THE RELATED ART

The growth of plants is influenced and decided by genetic features of plants and environmental factors like nutritional condition of soil , plant diseases , existence of favorable microorganism etc . There are soil bacteria, called plant growth-promoting rhizobacteria ( PGPR) , that promote plant growth in direct or indirect way .

Kloepper showed that the yields of seed potato treated with Pseudomonas putida, a plant growth- promoting bacterium, was increased by at least 10-15% or 40-70% occasionally, which research was performed repeatedly in several districts .

Suslow increased not only the yield of Beta vulgaris by utmost 32% but also the sugar yield per unit square by utmost 20% with use of plant growth- promoting bacteria .

Several methods have been known to promote plant growth directly with plant growth-promoting bacteria . There are such methods of promoting plant growth as using nitrogen fixation bacteria, supplying iron to plants by producing iron-siderophore , producing plant hormones like auxin, cytokinin and giberellin to promote plant growth, converting mineral not absorbable to plant like phosphorus to absorbable one, producing enzyme which enables to control generation of ethylene , a plant hormone .

It has been known that the method to promote plant growth by plant growth-promoting bacteria is indirectly controlling plant pathogen or harmful microorganism, or lowering the density of them.

The obj ect of the present invention is to provide plant growth-promotion factor derived from bacteria for promoting plant growth .

Pseudomonas fluorescens B16, a bacterium, in the present invention promotes the growth of tomato, pepper and so on in a new way different from known mechanisms . The pyrroloquinoline quinone ( PQQ) , which was reported lately as a new vitamin by the Rikkagakku research center in Japan, is a growth-promotion factor produced by Pseudomonas fluoresceins Bl6.

After PQQ was reported firstly as a cofactor of methanol dehydrogenase in methanolysis bacteria, it was also reported as a cofactor of alcoholysis enzyme, glucosidase, quinate dehydrogenase in bacteria like Klebsiella pneumoniae, Acinetobactor calcoaceticus and Methylobacterium organophilum etc . PQQ has been known to play an important role in mammals including human, but it has not yet known any role in plant growth . A research with mouse found that the deficiency of PQQ in mouse results in poor growth, and also makes the ability of breeding and immunity weak . It was also found that PQQ participates in degrading lysine, an essential amino acid, in mammals including human . That means that aminoadipic semialdehyde dehydrogenase (AASDH) gene has sequences binding with PQQ . If there was no PQQ, the said enzyme didn ' t work properly .

PQQ is water-soluble and has a similar chemical structure as vitamin B2 ( riboflavin) . Its chemical properties are similar to vitamin Bβ (pyridoxine) . Thus PQQ is classified as a vitamin B group . It is also known that PQQ exists at a small amount in plants like a parsley, green tea etc .

Once growth-promotion mechanism by bacteria is clarified, it is possible to promote plant growth and increase yield amounts and quality by means of activating related genes and entering an isolated growth-promotion factor gene into plants . Therefore, the research for bio-control agents applying such plant growth-promotion mechanism is being actively performed .

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other obj ects , features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings , in which;

FIG . 1 depicts a genetic map of PQQ biosynthesis region and phenotypes of each insertional mutation .

FIG . 2 depicts effects on the growth of tomatoes by the growth-promotion factor from Pseudomonas fl uorescens Blβ and its mutants .

FIG . 3 depicts the growth of tomatoes by the growth-promotion factor from Pseudomonas fluorescens Bl β and its mutant 818.

FIG . 4 depicts the ability of root colonization in tomatoes by Pseudomonas fluorescens B16 and its mutants .

FIG . 5 depicts the complementation of the mutant 818 by the growth-promotion factor from Pseudomonas fluorescens Blβ .

FIG . 6 shows the complementation result of the mutant 818 by growth-promotion factor from Pseudomonas fluorescens Blβ .

FIG . 7 depicts the result of above-ground length, diameter of stalk of tomato grown in the culture of nutrition broth for 2 years , which tomato contains growth-promotion factor from Pseudomonas fluorescens Blβ .

FIG . 8 depicts the result of the number of branches and flowers of tomato grown in the culture of nutrition broth for 2 years , which tomato contains growth-promotion factor from Pseudomonas fluorescens

Blβ . FIG . 9 depicts the results of the yield mass and weight of tomato containing growth-promotion factor from Pseudomonas fluorescens B16 in the culture of nutrition broth for 2 years .

FIG 10 shows HPLC result to identify growth- promotion factor from Pseudomonas fluorescens Blβ of the present invention .

DETAILED DESCRIPTION OF THIS INVENTION

The present invention relates to plant growth- promotion factor derived from Pseudomonas fluorescens Blβ and uses thereof, particularly to DNA molecules promoting plant growth, methods for promoting plant growth by applying the plant growth-promotion factor .

In addition, the present invention is to provide pyrroloquinoline quinone ( PQQ) , a plant growth- promoting factor for plant .

In addition, the present invention is to provide the recombinant vectors containing one or more DNA molecules of the present invention for transformation and gene expression, a transformed host having the said DNA molecule and a transformed plant containing the said DNA molecule . In addition, the present invention is to provide methods for promoting plant growth by using plant growth-promotion factor, which increases crop yield and improves its quality .

Hereinafter, the present invention will be described more clearly with accompanying drawings as follows .

The present inventors have isolated plant growth- promotion genes from Pseudomonas fluorescens Bl6 to accomplish the present invention . Those genes named pgpRABC and pqqABCDEFG participate in the plant growth .

The term "utilizing" in conj unction with the provision of PQQ refers to any conventional method to provide PQQ to plants . For example, the method includes transformation approaches using nucleotide sequences set forth in SEQ ID NOs : 1-11 , the introduction of proteins directly or indirectly into plants (or their tissues and organs) by using polypeptides set forth in

SEQ ID NOs : 12-22 and approaches to provide symbiotically by microbes transformed with nucleotide sequences set forth in SEQ ID NOs : 1-11. Fig . 1 shows the genome structure of Pseudomonas fluorescens B16 containing a new plant growth-promotion factor, wherein pOK40 is a cosmid clone and pOK53 is a pLAFR3 vector cloned only growth-promoting genome area into . The arrows depict genes and the numbers depict the mutants induced by transposon (Tn3-gusA) . The signs

( +) and ( - ) are used for showing whether the mutants promote plant growth and synthesize PQQ .

Base sequence of new growth-promoting factor in the present invention includes following sequence : pgpR gene ( SEQ ID NO : 1 ) , pgpA gene ( SEQ ID NO : 2 ) , pgpB gene ( SEQ ID NO : 3 ) , pgpC gene ( SEQ ID NO : 4 ) , pqqG gene ( SEQ ID NO : 5 ) , pqqE gene ( SEQ ID NO : 6 ) , pqqD gene ( SEQ ID NO : 7 ) , pqqC gene ( SEQ ID NO : 8 ) , pqqB gene ( SEQ ID NO : 9 ) , pqqA gene ( SEQ ID NO : 10 ) and pqqF gene ( SEQ ID NO : 11 ) .

In the present invention, the new plant growth- promotion factors include not only the said ' isolated ' DNA molecules but also DNA molecules that hybridize with DNA molecules having the said sequence list under stringent conditions and DNA molecules complementary to the hybridized DNA molecules .

The stringent condition, for example , is equivalent to over 60 ^° C for hybridization temperature or washing with 0.1 X concentration SSC for buffer solution .

DNA molecules , as a plant growth-promotion factor in the present invention, encode polypeptides (or proteins ) , of which amino acid sequences as follows :

PgpR polypeptide ( SEQ ID NO : 12 ) , PgpA polypeptide ( SEQ ID NO : 13 ) , PgpB polypeptide ( SEQ ID NO : 14 ) , PgpC polypeptide ( SEQ ID NO : 15 ) , PqqG polypeptide ( SEQ ID NO : 16 ) , PqqE polypeptide ( SEQ ID NO : 17 ) , PqqD polypeptide ( SEQ ID NO : 18 ) , PqqC polypeptide ( SEQ ID NO : 19 ) , PqqB polypeptide ( SEQ ID NO : 20 ) , PqqA polypeptide ( SEQ ID NO : 21 ) , PqqF polypeptide ( SEQ ID NO : 22 )

PQQ in the present invention, produced from Pseudomonas fluorescens B16, promotes plant growth .

PQQ is known to act as a cofactor of enzyme such as alcohol , glucose hydrolysis etc . in bacteria, however, it has not been reported that PQQ promotes plant growth well .

As described above, PQQ has been reported as a vitamin important in mammals, but there have been no reports about function in plants . In case that Pseudomonas fluoresceins B16 producing PQQ applied to plants , growth of plants was promoted to increase both yield mass and yield quality .

Adequately, the present invention can be applied to dicotyledones such as tomato, pepper, cucumber and paprika etc . and monocotyledones such as rice, corn, barley, wheat etc .

Therefore, the plant growth-promotion factor in the present invention can be applied to all plants .

The methods for promoting plant growth according to one aspect of the present invention include what apply plant growth-promotion factor from Pseudomonas fluorescens B16 to plants or plant seeds under such condition that plant growth-promotion factor reaches cell of plants or plant seeds .

The effect of growth-promotion according to the present invention presents diverse aspects such as early maturation, increase in yield mass etc .

In the present invention, plant growth-promotion factor and plant growth-promotion factor produced by Pseudomonas fluorescens B16 can be respectively applied to plant , but also can be applied as compositions having other additives .

The compositions having plant growth-promotion factor in the present invention may include fertilizers , pesticides , buffering agents, wetting agents and coating agents etc . The said plant growth-promoting compositions can be applied to plants or plant seeds by known methods such as spraying, wiping and immersing etc .

The isolated DNA molecules of the present invention can be transformed to cells by common DNA recombination technique . In plants , a plant body with propagation potential can re-differentiate from transformed single cell . Therefore, a gene transformed into plant cell can be inherited to next generation through seed.

In addition, the present invention pertains to expression vectors containing the isolated DNA molecules of the present invention . Diverse virus vectors and plasmid vectors can be used for the expression in the present invention . For instance , suitable expression vectors include pUC18 and pBluescript .

Host and vectors system for the expression of plant growth-promotion factor can be a transformed bacteria , microbe like yeast , virus , insect cell infected with virus and plant cell infected with bacteria . For specific example, E. coli-pBluescript host-vector system can be used .

The recombinant molecules in the present invention can be induced into plants by diverse methods . One of the methods for transforming the isolated DNA to plant cell is to use Ti plasmid . Agrobacterium tumefaciens, soil bacterium, contains giant plasmid (about 200 , 000 base pairs ) called Ti plasmid . When the said bacterium contacts with plant cell , T-DNA fragment transfers to plant cell nucleus from Ti plasmid and is integrated into chromosome during transformation . Similarly, Agrobacterium rhizogenes is also usable .

Another method for transforming the isolated DNA to plant cell is biolistics . During the transformation of plants by the biological ballistics , so called particle bombardment, target gene is entered to plant cell by shooting small metal particle containing or overlapped with foreign gene . The biological ballistics is useful in transformation of plants specially monocotyledones for which the transformation method using Agrobacterium does not work . The other method for inducing DNA is a microinj ection . The micro-inj ection is a method that minute amounts of material is inj ected into protoplast-like plant cell without cell wall through glass capillary tube which can be made with micro-manipulator on microscope .

Another method is to utilize liposome . When lipid solution is dropped in DNA or RNA aqueous solution, lipid vesicle ( liposome ) is formed to contain a little DNA or RNA aqueous solution by hydrophobic interaction between lipids .

The liposome containing DNA or RNA is so similar to protoplasmic membrane chemically that can be easily fused with protoplast and inj ected into specific internal cell organ ( i . e . vacuole ) by micro-inj ection .

Other methods for transforming the isolated DNA to plant cell are the using of electroporation and polyethylene glycol . The electroporation method enters a foreign DNA to protoplast by applying a strong and instant electric current into the DNA aqueous buffer solution having protoplasmic plant , as a result, foreign gene is entered to a chromosome of the plant . The polyethylene glycol ( PEG) has such strong absorptive power that high concentration of PEG solution transforms a cell membrane and makes DNA in PEG solution to move into cell .

After selection of the transformed cell by adding a selection marker such as antibiotics into the growth medium, the transformed protoplast is re-differentiated into plants .

While such re-differentiation methods are differ according to the species of plant , the known methods presented by Evans et al . in Handbook of Plant Cell ,

Vol . l (MacMillan Publishing Co . , New York, 1993 ) can be used .

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples .

However, it will be appreciated that those skilled in the art , on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention . <Example 1> Selection of mutant in promoting the growth of tomato

Pseudomonas fluσrescens B16 was conj ugated to E. coli S17-1 (pJFF350 ) , from which a large amount of mutant colony resistant to kanamycin antibiotics was selected . First of all, single colony of B16 mutant grown in LB plate was inoculated on 30ml of Agrobacterium minimal medium broth . The inoculated cell cultured in shaking incubator for 16 hours at 28 ° C with 230 rpm. After immersed in 30 ml of Blβ mutant culture solutions for an hour, tomato (cv . Kwangsu) seedling with four normal leaves was transplanted in square rockwool ( 10 x 10 x 7 cm) . Above-aerial part of the tomatoes was measured in length after 21 days from the day of transplantation to rockwool . With a repeated such experiment , mutant bacterium unable to promote growth was selected . Five mutants in growth-promotion were selected through fourth selection process , as shown in FIG . 2. Among them, mutant 818 showed the lowest ability on growth- promotion . Therefore, the mutant 818 was finally chosen . FIG . 3 shows that growth of tomato with mutant 818 is restricted .

<Example 2> Examination of root colonization by the strain Bl6 and its mutant in tomatoes Root colonization in tomatoes was surveyed 21 days after tomatoes treated with growth-promotion mutant were transplanted in square rockwool . One gram of tomato root was gathered from the rockwool , following by grinding thoroughly with 1 ml of steriled water in a mortar .

The grinded root was diluted adequately, and spread on LB medium containing 50 ug/ml of kanamycin .

After it was cultured in the nutrient medium for 24 hours in the constant temperature at 28 ^°C , the number of grown colonies was counted . FIG . 4 shows the bacterial population in the rhizosphere of five selected mutants . Among five mutants , bacterial population of only mutant 87 was 10 times lower than those of other mutants showing no changes in the

■ability of root colonization .

In case of mutant 818 that showed the lowest effect in growth-promotion, there were no changes in rhizosphere density. Therefore, it is ascertained that growth-promotion is not due to the lack of root colonization .

<Example 3> Complementation of growth-promotion mutant 818 Genome library of Blβ was manufactured . Gene fragment of growth-promotion mutant 818 was used as a probe to find a cosmid clone containing original genes from the genomic library of Blβ . This cosmid clone is named pOK40 , which complemented the mutant 818. As a control group, a vector (pLAFR3 ) enable to carry the recovered gene was put into mutant 818 in same conditions . After, tomatoes treated with the said mutants was transplanted in square rockwool , length of tomatoes above-aerial part was measured every three days from eighteenth day to thirty-third day after transplantation . FIG . 5 is a picture of the tomatoes 30 days after bacteria treatments . FIG . 6 depicts a diagram that shows the result of the change in the length of tomatoes above-aerial part . As shown in FIG . 5 and FIG . 6, the effect of growth-promotion provided by mutant 818 was considerably low; compared to the effect of growth-promotion by Blβ, and the mutant 818 carrying the cosmid clone (pOK40 ) regained its ability to promote growth . The mutant 818 containing vector only, as a control group, showed no improvement in growth-promotion . Therefore , it is ascertained that the gene providing growth-promoting is located in cosmid clone, pOK40.

<Example 4> Growth-promotion effect on tomato in the system of nutrient solution

B16, mutant 818 and mutant 818 carrying pOK40 had been cultured in Agrobacterium medium at 28 ^°C for 36 hours . Tomatoes with six normal leaves were immersed for an hour with bacteria suspension which has 10⁸ cfu/ml bacterial concentrations in sterilized water . The said bacteria-treated tomatoes were transplanted in culture system of nutrition broth and the remained bacterial suspension was poured into the plants . The bacterial solution of 100ml made by the above method was drenched with ten-day interval , seven times , after the transplantation . After 21 days from the transplantation, growth of tomato ( i . e . length above- ground, diameter of stalk, the number of branches , number of flowers ) , and the number of fruits were surveyed.

FIG . 7 is shows the change in length above-ground and diameter of stalk in tomatoes . FIG . 8 shows the change in number of branches and flower of tomatoes .

FIG . 9 shows the change of mass and weight of harvested tomatoes .

It is concluded that B16 promoted the length of above-ground part , number of branches , diameter of stalk and even mass yield of tomatoes , but mutant 818 did not promote growth of tomatoes . The mutant 818 complemented with pOK40 promoted growth of tomatoes . Therefore, it is ascertained that the mutated region in mutant 818 is the portion that promote the growth of plants .

<Example 5> Identification of growth-promotion factor

Pseudomonas fluorescens B16 was cultured at 28 °C for 48 hours in Agrobacterium minimal medium added with 0.4% gluconic acid as carbon source . After nine times volume of methanol was added into bacterial suspension culture, it was evaporated in distillation apparatus . The said distillate was solved with 70% methanol , and was passed through Sep-Pak (C18 column) . Then, the final material solved with 70% methanol was mixed with Na₂B₄O₇ of 0.2 M, set to pH 8.0 , and reacted with 0.5% acetone . FIG . 10 shows the results of the reverse phase HPLC analysis of the said reacted solution . When PQQ is reacted with acetone , acetone combines to PQQ, called 5-acetonyl PQQ . As shown in FIG 10 , peaks of material produced by Pseudomonas fluorescens Blβ are in same position with those of synthesized PQQ . In mutant k433 which lost ability to promote plant growth due to insertion of transposon (Tn3-gusA) into PQQ gene, PQQ peak did not appear (Ref . FIG 1 ) . Mutant K433 carrying pOK53 regained ability to synthesize PQQ . Therefore, the results show that plant growth-promotion factor from Pseudomonas fluorescens Bl 6 is PQQ .

As described and confirmed above , the plant growth-promotion factor and its derivatives of the present invention can be applied to plants or plant seeds widely so as to promote the plant growth highly and to increase product yield industrially .

In addition, the transformed plants that are prepared by the method of the present invention, using the plant growth-promotion DNA and the resulting plants that are grown from the transformed seeds are useful to provide more excellent rate of growth .

Claims

What is claimed is :

1. A method for promoting plant growth, by providing a plant with pyrroloquinoline quinone ( PQQ ) .

2. The method for promoting plant growth according to claim 1 , wherein PQQ is provided to the plant by utili zing a DNA molecule comprising any one of the nucleotide sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO : 11.

3. The method for promoting plant growth according to claim 1 , wherein PQQ is provided to the plant by utilizing a polypeptide comprising any one of amino acid sequences selected from the group consisting of SEQ ID NO : 12 to SEQ ID NO : 22.

4. The method for promoting plant growth according to claim 1 , wherein PQQ is provided to the plant by utilizing one or more nucleotide sequences selected from the group consisting of the following sequences :

(a ) a nucleotide sequence comprising any one of nucleotide sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO : 11 ; (b) a nucleotide sequence hybridi zing to a nucleotide sequence comprising any one of nucleotide sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO : 11 under stringent conditions of hybridization; and

( c) a complementary nucleotide sequence hybridizing to said nucleotide sequence of (b) under stringent conditions of hybridization .

5. A nucleotide sequence for promoting plant growth, which is one or more nucleotide sequences selected from the group consisting of the following sequences :

(a) a nucleotide sequence comprising any^¬ one of nucleotide sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO : 11 ;

(b) a nucleotide sequence hybridizing to a nucleotide sequence comprising any one of nucleotide sequences selected from the group consisting of SEQ ID NO : 1 to SEQ ID NO : 11 under stringent conditions of hybridization; and

(c) a complementary nucleotide sequence hybridizing to said nucleotide sequence of (b) under stringent conditions of hybridization .

6. A polypeptide for promoting plant growth, which has one or more amino acid sequences selected from the group consisting of SEQ ID NO : 12 to SEQ ID NO : 22.

7. A recombinant vector, which comprises the nucleotide sequence of claim 5.

8. A plant cell , which is transformed with the nucleotide sequence of claim 5.

9. A callus , which is prepared by using the plant cell of claim 8.

10. A plant , which is transformed with the nucleotide sequence of claim 5.

11. A plant seed, which is obtained from the plant of claim 10.