U.S. patent application number 10/224567 was filed with the patent office on 2003-05-08 for method to promote growth of a plant.
Invention is credited to Fukusaki, Eiichiro, Isogai, Akira, Kobayashi, Akio.
Application Number | 20030087763 10/224567 |
Document ID | / |
Family ID | 26397772 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087763 |
Kind Code |
A1 |
Kobayashi, Akio ; et
al. |
May 8, 2003 |
Method to promote growth of a plant
Abstract
A concise method to promote growth of a plant useful for
increase of food production. This invention provides a method to
promote growth of a plant by administration of formic acid, an
organic acid with a low molecular weight through avoidance of
photo-oxidation damage. This invention also provides amino acid
sequence of formate dehydrogenase and base sequence encoding the
enzyme. The expression of the enzyme is induced by administration
of formic acid and the enzyme is involved in metabolism, of formic
acid and its salts. Furthermore, this invention provides a method
to promote growth of a plant by administration of methanol through
avoidance of photo-oxidation damage.
Inventors: |
Kobayashi, Akio; (Osaka,
JP) ; Fukusaki, Eiichiro; (Osaka, JP) ;
Isogai, Akira; (Ikoma City, JP) |
Correspondence
Address: |
Robert G. Mukai
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
26397772 |
Appl. No.: |
10/224567 |
Filed: |
August 21, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10224567 |
Aug 21, 2002 |
|
|
|
09517427 |
Mar 2, 2000 |
|
|
|
6465396 |
|
|
|
|
Current U.S.
Class: |
504/320 ;
435/189 |
Current CPC
Class: |
A01N 37/02 20130101;
A01N 31/02 20130101 |
Class at
Publication: |
504/320 ;
435/189 |
International
Class: |
A01N 037/00; A01N
031/00; C12N 009/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 1999 |
JP |
11-56,776 |
Feb 15, 2000 |
JP |
2000-36,153 |
Claims
What is claimed is:
1. A method to promote growth of a plant, the method comprising
administration of an organic acid with a low molecular weight or
its salt to a plant.
2. The method according to claim 1, the method comprising
administration of the organic acid or its salt to avoid
photo-oxidation damage of the plant.
3. The method according to claim 1, wherein said organic acid or
its salt is formic acid or a formate salt.
4. The method according to claim 3, wherein said formate salt is
selected from the group consisted of potassium formate, sodium
formate and ammonium formate.
5. The method according to claim 3, the method comprising
administration of the organic acid or its salt to induce enzyme
activity of formate dehydrogenase.
6. A plant yield promoting agent, the agent comprising an organic
acid with a low molecular weight or its salt.
7. The plant yield promoting agent according to claim 6, wherein
said organic acid is formic acid or a formate salt.
8. The plant yield promoting agent according to claim 7, the agent
containing formic acid or a formate salt at the concentration of
125 ppm.
9. The plant yield promoting agent according to claim 7, wherein
the agent contains formic acid or a formate salt and has a pH
adjusted to 5.5-8.0.
10. The plant yield promoting agent according to claim 7, wherein
said formate salt is selected from the group consisted of potassium
formate, sodium formate and ammonium formate.
11. A peptide fragment derived from rice formate dehydrogenase
having an amino acid sequence of following (a) or (b): (a) an amino
acid sequence referred to as amino acid numbers from 1 to 376 in
sequence number 1 in a sequence list, (b) an amino acid sequence in
which a part of said amino acid sequence (a) is deleted or another
amino acid sequence is added to said amino acid sequence (a) or a
part of said amino acid sequence (a) is substituted with another
amino acid sequence, the amino acid sequence (b) exhibiting enzyme
activity to synthesize carbon dioxide using formic acid as
substrate.
12. A DNA fragment derived from rice formate dehydrogenase having a
base sequence of following (c) or (d): (c) a base sequence referred
to as base numbers from 1 to 1450 in sequence number 2 in a
sequence list, (d) a base sequence hybridizes with said base
sequence (c) under stringent condition, the base sequence (d)
encoding a protein exhibiting enzyme activity to synthesize carbon
dioxide using formic acid as substrate.
13. A method to promote growth of a plant, the method comprising
incorporation of DNA fragment according to claim 12 into a plant
and over-expression of formate dehydrogenase in the plant.
14. A transgenic plant produced by incorporation of DNA fragment
according to claim 12 into a plant and over-expression of formate
dehydrogenase in the plant.
15. A method to promote growth of a plant, the method comprising
administration of an alcohol with a low molecular weight to the
plant.
16. The method according to claim 15, wherein said alcohol is
methanol.
17. The method according to claim 15, the method comprising
administration of the alcohol to avoid photo-oxidation damage of
the plant.
18. A plant yield promoting agent containing methanol.
19. The plant yield promoting agent according to claim 18, the
agent containing methanol at a concentration of 50 mM
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a method to promote growth of a
plant by administration of an organic acid with a low molecular
weight to avoid photo-oxidation damage of the plant. This invention
also relates to a method to promote growth of a plant by
administration of an alcohol to avoid photo-oxidation damage of the
plant.
[0003] 2. Description of Related Art
[0004] Nowadays, shortage of food production have became a serious
problem because of population explosion throughout the world.
Growth and metabolism of a plant are affected by various conditions
of surroundings. Photo-oxidation damage is caused by alteration of
surrounding conditions and triggered by decrease of carbon dioxide
uptake, through closure of stoma under condition of strong
light-intensity and droughtiness. Because of shortage of carbon
dioxide under such condition, excess amount of energy is acquired
by photosynthesis system. As the result, excess amount of active
oxygen beyond defense ability of the plant is generated, which
results in occurrence of the photo-oxidation damage. As such
photo-oxidation damage inhibits plant growth, a method to prevent
this phenomenon is desired. Heretofore, a transgenic plant having
ability to delete active oxygen have been used for such
purpose.
SUMMARY OF THE INVENTION
[0005] Despite of it, production of a transgenic plant with such
characteristic takes much time and the range of plants suitable for
producing such transgenic plant is limited. Therefore, the
development of a method to prevent photo-oxidation damage with
simplicity and wide-utility have been desired to promote growth of
a plant.
[0006] It is known that, a plant metabolizes methanol and converts
it to carbon dioxide, through formaldehyde and formic acid as the
intermediate. Then the inventors administrated formic acid, an
organic acid with a low molecular weight, to rice, tabacco and
kidney bean and found avoidance of photo-oxidation damage.
Moreover, the inventors have investigated effect of formic acid on
growth of rice plant bodies and found its effectiveness on growth
promotion. Moreover, the induction of formate dehydrogenase (FDH)
activity, which metabolizes formic acid to produce carbon dioxide,
was observed by administration of formic acid. Furthermore, the
amino acid sequence of formate dehydrogenase (FDH) and the base
sequence encoding the enzyme were determined. The photo-oxidation
damage might be prevented by incorporation of FDH gene.
[0007] The inventors have also administrated methanol, an alcohol
with a low molecular weight, to avoid photo-oxidation damage of
rice, tabacco and kidney bean. Therefore, methanol might be
effective for promotion of plant growth.
[0008] These and other features and advantages of this invention
will become apparent upon a reading of the detailed description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a graph showing effects of various formate salts
on photo-oxidation damage of rice.
[0010] FIG. 2 is a graph showing time course of photo-oxidation
damage of rice and inhibition of photo-oxidation damage by
potassium formate.
[0011] FIG. 3 is a graph showing effect of formic acid on
photo-synthetic ability of rice under various intensities of
illumination after photo-oxidation damage.
[0012] FIG. 4 is a graph showing dose-dependency of potassium
formate concentration on inhibition of photo-oxidation damage of
rice.
[0013] FIG. 5 is a graph showing inhibition of photo-oxidation
damage of kidney bean by formate salts.
[0014] FIG. 6 is a graph showing inhibition of photo-oxidation
damage of tabacco by formate salts.
[0015] FIG. 7 is a graph showing result of fluorescence analysis of
rice after photo-oxidation damage.
[0016] FIG. 8 is a graph showing dose dependency of sodium formate
concentration on wet weight of rice plant bodies.
[0017] FIG. 9 is a graph showing dose dependency of sodium formate
concentration on height of aerial part of rice plant bodies.
[0018] FIG. 10 is a graph showing time course of formic acid uptake
measured on rice.
[0019] FIG. 11 is a graph showing time course of carbon dioxide
formation originated from formic acid measured on rice.
[0020] FIG. 12 is a graph showing activity of formate dehydrogenase
measured on aerial part and ground part of rice plant bodies.
[0021] FIG. 13 is a graph showing amino acid sequence of rice
formate dehydrogenase, compared with that of potato formate
dehydrogenase and barley formate dehydrogenase.
[0022] FIG. 14 is a photograph of northern blotting, indicating
alteration of FDH gene expression by administration of formic
acid.
[0023] FIG. 15 is a graph showing effect of methanol and potassium
formate on photo-oxidation damage of rice.
[0024] FIG. 16 is a graph showing effect of methanol on
photosynthetic ability of rice under various intensities of light
illumination.
[0025] FIG. 17 is a graph showing dose-dependency of methanol on
inhibition of photo-oxidation damage of rice measured by
fluorescence analysis.
[0026] FIG. 18 is a graph showing effect of methanol and formic
acid on tabacco, measured by fluorescence analysis.
[0027] FIG. 19 is a graph showing effect of methanol and formic
acid on kidney bean, measured by fluorescence analysis.
DETAILED DESCRIPTION OF EMBODIMENTS
[0028] (The Plant Bodies Utilized for the Experiments)
[0029] (1) Rice
[0030] The seeds of rice (Oryza sativa L.: Hinihikari) were used
for the experiments. The plants were seeded under a vermiculight.
They were grown under a light intensity of 150 .mu.mol
quanta/m.sup.2.s for 16 hours in the light and 8 hours in the dark.
Normal leaves derived from the plant bodies of 2 to 4 weeks old
were used for the following experiments.
[0031] (2) Tabacco
[0032] The seeds of tabacco (Nicotina tabacum L.: SRI) were used
for the experiment. The plants were seeded under a vermiculight.
They were grown under the light intensity of 150 .mu.mol
quanta/m.sup.2.multidot.s. Normal leaves derived from the plant
bodies of 4 to 6 weeks old were used for the experiments.
[0033] (3) Kidney Bean
[0034] The seeds of kidney bean (Phaseolus vulgaris L.:
tsurunashiinngenn) were used for the experiments. The plants were
seeded under a vermiculight. They were grown under the light
intensity of 150 .mu.mol quanta/m.sup.2.multidot.s. Normal leaves
derived from the plant bodies of 2 to 4 weeks old were used for the
experiments.
[0035] (Method for Administration of Sample Compounds)
[0036] In the case of rice plant, a portion containing petiole was
excised from the plant bodies and it was immersed into 10 ml of
sample compound in a tube. It was incubated for 30 min at
25.degree. C. under 150 .mu.mol quanta/m.sup.2.multidot.s of
illumination. After administration of sample compounds, the leaves
of rice were sliced and put in a chamber with diameter of 35 mm and
the rate of oxygen generation (.mu.mol O.sub.2/m.sup.2.multidot.s)
was estimated. In the case of other plants, a leaf disk with
diameter of 35 mm was prepared prior to the experiment. It was
immersed into 10 ml of sample solution in a dish and the sample
solution was administrated under the same condition as rice plant.
After the administration, water in the leaf disk was excluded, the
leaf was set in the chamber and the ability of photosynthesis was
measured on the leaf. Then, the sample solution was prepared by
dissolving arbitrary amount of sample compounds into 10 mM
potassium phosphate buffer (pH7) and sample solution thus prepared
was used for the experiments. Potassium formate, sodium formate and
ammonium formate were used for the sample compounds.
[0037] (Estimation of Photosynthetic Ability in Terms of Oxygen
Generation Rate)
[0038] Generation of oxygen was measured by clark type oxygen
electrode. The cuvette (LD2/2 type), the oxygen electrode
controller (CB1-D3 type) and the light source (LS-2, Hansatec
corp.) were adopted as components of the measuring device. The
chamber of cuvette contained in this device is composed of
combination of a chamber, capable of hermetically sealing with
diameter of 35 mm and volume of 5 ml, and an oxygen electrode. As
upper side of the chamber is transparent, arbitrary intensity of
light can be illuminated. The plant body described above, after
administration of formic acid, was enclosed in the chamber and
sealed hermetically. It was illuminated using a halogen lamp as the
light source, and the rate of oxygen generation was measured under
the air containing 5% of carbon dioxide. Unless specified
otherwise, the measurement was performed under the light adjusted
to a intensity of 253 .mu.mol quanta/m.sup.2.multidot.- s using a
red filter or an attenuation filter. The oxygen generation was
calculated by dividing the obtained value with the surface area of
the leaf.
[0039] (The Condition of Photo-oxidation Damage)
[0040] After measurement of rate of photosynthesis as described
above, the air without carbon dioxide was introduced into the
chamber and the light was illuminated at the intensity of 1600
.mu.mol quanta/m.sup.2.multidot.- s for a certain period. The
photo-oxidation damage, caused by lack of carbon dioxide, was
induced under the condition described above. The rate of oxygen
generation was measured after this treatment and inhibition of
photosynthetic ability caused by photo-oxidation damage was thus
evaluated.
[0041] (The Effect of Formate Salts on Photo-oxidation Damage)
[0042] The effect of various formate salts on the photo-oxidation
damage of rice was investigated. The rate of oxygen generation was
reduced to about 30% by 8 min of strong light illumination in the
absence of carbon dioxide as shown in FIG. 1. On the other hand,
plant bodies retained 90% of oxygen generation activity by addition
of 2 mM of potassium formate. Such protection effect was the most
prominent on potassium formate. Two mM of formate salts exhibited
potency on protection of oxygen generation ability in the turn of
potassium formate, sodium formate and ammonium formate. Moreover,
time course of protection of photo-oxidation damage by formate
salts was investigated. Prolonged treatment (2 min, 4 min) of
photo-oxidation damage caused damage on photosynthetic ability in a
time dependent manner as shown in FIG. 2. Despite of it, the damage
on photosynthetic ability was inhibited in the presence of 2 mM
potassium formate.
[0043] Furthermore, the effect of light intensity on photosynthetic
ability of rice sample was investigated after photo-oxidation
damage treatment. As shown in FIG. 3, the inclination was not
altered under weak light illumination, whereas the addition of
formate salt inhibited damage on photosynthetic ability under
strong light illumination. From this result, enzyme system
concerning calvin cycle might be protected in the presence of
formate salts. Moreover, the dose-dependency of potassium formate
concentration on protection of photo-oxidation damage, caused by
potassium formate, was investigated on rice. As the result, 2 mM or
4 mM of potassium formate exhibited optimum protection on
photo-oxidation damage as shown in FIG. 4.
[0044] The effect of formate salts on photo-oxidation damage was
also observed on kidney bean and tabacco. Administration of 2 mM of
potassium formate exhibited inhibition on oxygen generation rate
caused by photo-oxidation damage. FIG. 5 shows the result on kidney
bean and FIG. 6 shows the result on tabacco plant.
[0045] (Fluorescence Measurement of Chlorophyl)
[0046] To estimate sanity of photo-system, the fluorescence
originated from chlorophyl, the center of photo-system, was
measured on rice under the same condition described above after
photo-oxidation damage. The measurement was performed by chlorophyl
fluorometer (PAM101: emitter and detector, PAM103: light pulse
generator, WALTZ). The condition of measurement was as follows.
Excitation: 100 KHz, intensity: 5, saturated light: 900 ms. The
fluorescence was measured before and after photo-oxidation damage
to estimate Fv/Fm value. The condition of plant growth, the method
to administrate formic acid and the preparation of samples adopted
in this experiment were the same as the experiments on oxygen
generation. The result of fluorescence measurement revealed that
damage on photo-system, caused by photo-oxidation damage, was
avoided by addition of 2 mM formate (FIG. 7).
[0047] (Plant Growth Test)
[0048] The effect of formate salts addition observed on plant
growth was investigated. The seeds of rice were sterilized and
seeded into a glass tube (130 mm.times.40 mm I.D) containing medium
described below. The contents of the medium are 1/2 MS salt, 0.1%
gellan gum and various concentrations of sodium formate. The pH was
adjusted to 6.0 by addition of 1N sodium hydroxide and the medium
was sterilized by autoclave treatment. The rice seeds were grown
under 25.degree. C. for 16 hours in the light and 8 hours in the
dark, under 40 .mu.mol quanta/m.sup.2.multidot.s of illumination.
After cultivation for 2 weeks under this condition, the extent of
growth was estimated by measurement of length of the aerial part
and wet weight of plant bodies.
[0049] The effect of formate salts on growth of rice plant bodies,
measured as described above, was investigated. The effect of sodium
formate was investigated under various concentrations to reveal
that, addition of 125 ppm of sodium formate resulted in 25% of
growth promotion, both on wet weight of plant bodies (FIG. 8) and
length of aerial part (FIG. 9). The growth promotion effect
disappeared at the presence of 1000 ppm of sodium formate. The
student t-test was performed on difference of average value
calculated on 30 samples. The result showed significant difference
at a significantly low level of less than 1%. Therefore, the
formulation containing 125 ppm of sodium formate with pH adjusted
to pH7 is effective for promotion of plant growth. Formic acid, the
effective ingredient of this formulation, can be added to ordinal
phosphate fertilizer or nitrogen fertilizer. As an example of such
fertilizer, a commercial fertilizer "hyponex" is preferred.
[0050] (Uptake of Formic Acid into Plant Bodies)
[0051] To confirm metabolism of formic acid, uptake of formic acid
into plant body was investigated under the condition described at
"plant growth test", using rice cultivated for 2 weeks at
25.degree. C. Five plant bodies were harvested and washed by water,
then 2.5 ml of 1.4 mM sodium formate was added and incubated under
illuminated condition (40 .mu.mol quanta/m.sup.2.multidot.s) at
25.degree. C. Twenty-five .mu.l of the solution was sampled at each
period and formic acid concentration in the solution was determined
by absorbance. The result exhibited rapid uptake of formic acid and
formic acid was not detected in the solution after 48 hours. FIG.
10 shows the result as the average value of three independent
experiments. Formic acid is assumed to be absorbed in the plant
body because decrease of formic acid was not observed without the
plant body (control).
[0052] (Formation of Carbon Dioxide by Administration of Formic
Acid)
[0053] The extent of carbon dioxide produced by metabolism of
formic acid was measured using rice plant bodies cultivated at
25.degree. C. for two weeks as described above. The glass container
containing 5 ml of 1.4 mM [.sup.12C] or [.sup.13C] sodium formate
(pH7) was sealed by gumseptam and incubated at 25.degree. C. under
dark condition. Twenty-five .mu.l of the solution was sampled at
each period and content of carbon dioxide in the supernatant was
analyzed by gas chromatography-mass spectrometry (GC-MS). As the
result, 9% (FIG. 11A) or 1% (FIG. 11B) of [.sup.13C] CO.sub.2,
compared to total carbon dioxide, was detected in samples with
addition of [.sup.12C] or [.sup.13C] formic acid, respectively, as
shown in FIG. 11. As natural content of [.sup.13C] is about 1%,
increased [.sup.13C] CO.sub.2 would be originated from added formic
acid. This result indicates existence of certain mechanism that
converts formic acid to carbon dioxide in rice, for example,
formate dehydrogenase (FDH).
[0054] (Measurement of FDH Activity)
[0055] Then the alteration of FDH activity by administration of
formic acid was investigated. The rice plant bodies were cultivated
in the presence or absence of 1.4 mM formic acid at 25.degree. C.
for two weeks. The aerial part and the ground part (root) was
separated and the crude enzyme solution was prepared from each
part. The enzyme solution was prepared to 1.5 mg/ml and FDH
activities of the samples were measured. The FDH activity was
measured using [.sup.13C] formic acid as the substrate and
detecting [.sup.13C] CO.sub.2 in the supernatant by GC-MS. When the
root of rice was cultivated in the presence of formic acid, its
activity was about twice compared to that of control group (without
addition of formic acid), indicating increase of enzyme activity
(FIG. 12).
[0056] (Isolation of RNA)
[0057] Isolation of total RNA was performed as described below. In
the case of utilization for northern analysis, total RNA was
isolated from leaf and root of rice cultivated under condition
described above, using Isogen RNA purification kit (Nippon gene).
In the case of cDNA preparation, total RNA was isolated using Plant
RNA kit (QIAGEN). After treatment by DNAase, oligo(dT) 12-18 primer
(GIBCOBRL) was used for reverse-transcription and cDNA was
prepared.
[0058] (Cloning of cDNA Encoding FDH)
[0059] From sequence homology with FDH derived from potato, three
clones of rice cDNA were selected from DDBJ (DDBJ accession nos.
D23989, D23770 and D48722). The alignment of these cDNA clones
revealed that, these cDNA sequences are constituting partial
sequence of FDH homolog consisted of 970 bp. Then this partial
sequence was cloned from rice cDNA using PCR. Using this sequence,
full length of FDH gene was isolated using 3'- and 5'-Full Race
core Set (Takara biochemicals).
[0060] (Determination and Analysis of the Base Sequence)
[0061] PCR products were subcloned to PCR2.1 (Invitrogen) or pUC18
(Takara biochemicals). The sequence of subcloned product was
sequenced by DNA sequencer (ABI 310, Applied Biosystems), using M13
primer and sequencing kit (dye terminator cycle sequencing ready
reaction kit, Perkin Elmer). The result showed that, the gene
consists of a base sequence with reading frame of 1450 base pair
(sequence list: sequence number 2). The sequence was analyzed using
genetic-mac (genetic information processing software), a sequence
analysis software. The result revealed that the deduced amino acid
sequence of FDH consists of 376 base pair (sequence list: sequence
number 1), with calculated molecular weight of 41.2 kDa and
isoelectric point of 7.39. The sequence homology was compared
between this sequence and FDH sequences originated from other known
plants. The result exhibited high homology with known FDH sequences
of other plants. That is, the amino acid sequence of rice
(C.sativa) FDH exhibited high homology with potato (S.tuberosum:
82.7%) FDH and barley (H.vulgare: 91.7%) FDH. Moreover, the formic
acid binding site (282) and the NAD binding site (189-223) were
shown to be highly conserved among these plants (FIG. 13). As amino
acid sequence of rice FDH and base sequence encoding the enzyme
were determined, resistance against photo-oxidation damage might be
rendered to a plant by incorporation of the gene described in
sequence number 2 in the sequence list.
[0062] (Expression of cDNA in E.coli.)
[0063] The amino acid sequence deduced from the gene obtained by
the cloning described above showed high homology with known FDH.
Therefore, this sequence was expected to encodes FDH and the cDNA
was expressed in E.coli to confirm the function of this sequence.
The plasmid, utilised to be expressed in E.coli, was prepared by
insertion of target fragment into pUC18 plasmid abscission. The
fragment was prepared by PCR amplification using primers containing
restriction enzyme recognition sites and then the product was
treated by restriction enzyme. The fragment thus prepared was
inserted into pUC18 plasmid. The primer containing EcoRI site and
BamHI site was prepared in two series and the insertion fragments
were prepared for two orientations. That is, insertion orientations
of the sequence are sense orientation and anti-sense orientation.
The primers used were 5'-cggaattcatggcgatgtggagggcggc-3' (forward
direction) and 5'-cgggatccttactggtactggc-3' (reverse direction) for
the sense orientation and 5'-cgggatccatggcgatgtggagggcg-3' (forward
direction) and 5'-cggaattcttactggtactggctcgcgagc-3' (reverse
direction) for the anti-sense orientation (Reverse). The PCR
products using the primers were incorporated into pUC18 plasmid by
EcoRI site and the BamHI site, to the initiation codon or the
termination codon. The insertion of cloned sequence was confirmed
by sequencing using di-deoxy method utilizing M13 primer.
[0064] The E.coli JM109 strain was transformed by the plasmid thus
prepared. Transformed E.coli was cultured in 50 ml of LB medium
(containing 50 mg/ml ampicillin) until the absorbance at 660 nm
reached to 3.0. It was cultured under aerobic condition at
temperature of 20.degree. C., which is a lower temperature compared
to ordinary condition. Then expression of protein was induced by
0.2 mM IPTG at 20.degree. C. for 6 hours. The bacteria was
collected, then washed by water and re-suspended into 2 ml of
solution containing 10 mM SPB (pH 7.0), 100 mM NaCl, 0.5% triton
X-100 and 2 mM DTT. The bacteria obtained was disrupted by
sonication and the crude enzyme solution was prepared from
supernatant of the centrifugation. The centrifugation was performed
at 15,000 g for 15 min and the supernatant was obtained to measure
enzyme activity. The intrinsic FDH of E.coli does not utilize NADH
as a co-enzyme, as described in the report, the protein did not
exhibit enzyme activity at the actual measurement. It was also
revealed that, only the fragments with sense-orientated insertion
exhibited enzyme activity. Therefore, it was strongly suggested
that the sequence would be FDH protein with dependency to NAD.
[0065] (Alteration of FDH Gene Expression by Administration of
Formic Acid)
[0066] The enzyme activity of rice FDH increased by administration
of formic acid. Therefore, the expression of FDH gene, isolated in
this experiment, was analyzed. Like measurement of enzyme activity,
the rice plant was cultivated with or without formic acid. RNA was
prepared from the aerial part and the ground part (root)
respectively and the northern analysis was performed on these
samples. Forty .mu.g of the total RNA was applied to each lane and
electrophoresis was performed on 1% agarose gel (containing 2.2 M
formaldehyde). The gel was charged for the capillary blotting using
Hybond N+ membrane (Amersham) using 20.times.SSC. The hybridization
was performed according to instruction of AlkPhosDIREST (Amersham)
and detection was performed using CDP-Star (Amersham). The FDH cDNA
(open reading frame: 1130 bp), amplified by PCR, was used as probe
DNA. The expression of FDH gene, measured on ground part of rice,
exhibited significant increase by pretreatment with formic acid
administration (FIG. 14).
[0067] As described above, a metabolite pathway described below
exists in some plant species including rice. That is, methanol is
metabolized to carbon dioxide by oxidation in a stepwise manner,
through conversion to formaldehyde and formic acid as the
intermediates. Then methanol, an alcohol with a low molecular
weight, might be effective for inhibition of photo-oxidation damage
and investigation was performed on the hypothesis. The plant bodies
used in the experiment, the method to administrate sample
substance, evaluation of photosynthesis by measurement of oxygen
generation rate, photo-oxidation damage measurement and chlorophyl
fluorescent measurement were performed as described in the
experiments of formic acid.
[0068] (Effect of Methanol on Photosynthetic Ability of Rice)
[0069] By analysis of oxygen generation rate, effect of methanol on
photosynthetic ability was investigated. The result indicated that,
when oxygen generation rate of the control sample was reduced to
30%, the residual photosynthetic ability was 70% and 60% by
addition of methanol and formate salt, respectively (FIG. 15). The
dependency of light intensity was investigated after
photo-oxidation damage of rice, using oxygen generation rate as an
indicator (FIG. 16). The administration of methanol avoided
photo-oxidation damage as shown in FIG. 16, especially under strong
light illumination.
[0070] (Effect of Methanol on Chlorophyl Fluorescence Analysis of
Rice Plant)
[0071] Furthermore, chlorophyl fluorescence analysis was performed
under presence of various concentrations of methanol using rice.
The addition of 25 mM or 50 mM methanol resulted in significant
inhibition of reduction of Fv/Fm value caused by photo-oxidation
damage (FIG. 17).
[0072] (Effect of Methanol on Chlorophyl Fluorescence Analysis of
Tabacco and Kidney Bean)
[0073] Furthermore, the effect of methanol addition was also
investigated on tabacco (FIG. 18) and kidney bean (FIG. 19). The
chlorophyl fluorescence analysis revealed potency of 100 mM
methanol addition, as the same as 2 mM formic acid addition. Then
the damage on photo-system, caused by photo-oxidation damage, was
significantly alleviated by addition of methanol.
[0074] A method to promote plant growth was proposed by this
invention. It was achieved by administration of formic acid, an
organic acid of low molecular weight, through avoidance of
photo-oxidation damage. Moreover, amino acid sequence of formate
dehydrogenase and base sequence encoding the enzyme were also
proposed. In addition, a method to promote plant growth by
administration of methanol, through avoidance of photo-oxidation
damage, was proposed.
Sequence CWU 1
1
2 1 376 PRT Rice formate dehydrogenase VARIANT 287 Xaa = Any Amino
Acid 1 Met Ala Met Trp Arg Ala Ala Ala Gly His Leu Leu Gly Arg Ala
Leu 1 5 10 15 Gly Ser Arg Ala Ala His Thr Ser Ala Gly Ser Lys Lys
Ile Val Gly 20 25 30 Val Phe Tyr Lys Gly Gly Glu Tyr Ala Asp Lys
Asn Pro Asn Phe Val 35 40 45 Gly Cys Val Glu Gly Ala Leu Gly Ile
Arg Glu Trp Leu Glu Ser Lys 50 55 60 Gly His His Tyr Ile Val Thr
Asp Asp Lys Glu Gly Leu Asn Ser Glu 65 70 75 80 Leu Glu Lys His Ile
Glu Asp Met His Val Leu Ile Thr Thr Pro Phe 85 90 95 His Pro Ala
Tyr Val Ser Ala Glu Arg Ile Lys Lys Ala Lys Asn Leu 100 105 110 Glu
Leu Leu Leu Thr Ala Gly Ile Gly Ser Asp His Ile Asp Leu Pro 115 120
125 Ala Ala Ala Ala Ala Gly Leu Thr Val Ala Glu Val Thr Gly Ser Asn
130 135 140 Thr Val Ser Val Ala Glu Asp Glu Leu Met Arg Ile Leu Ile
Leu Leu 145 150 155 160 Arg Asn Phe Leu Pro Gly Tyr Gln Gln Val Val
His Gly Glu Trp Asn 165 170 175 Val Ala Gly Ile Ala Tyr Arg Ala Tyr
Asp Leu Glu Gly Lys Thr Val 180 185 190 Gly Thr Val Gly Ala Gly Arg
Ile Gly Arg Leu Leu Leu Gln Arg Leu 195 200 205 Lys Pro Phe Asn Cys
Asn Leu Leu Tyr His Asp Arg Leu Lys Ile Asp 210 215 220 Pro Glu Leu
Glu Lys Glu Ile Gly Ala Lys Tyr Glu Glu Asp Leu Asp 225 230 235 240
Ala Met Leu Pro Lys Cys Asp Val Ile Val Ile Asn Thr Pro Leu Thr 245
250 255 Glu Lys Thr Arg Gly Met Phe Asn Lys Glu Arg Ile Ala Lys Met
Lys 260 265 270 Lys Gly Val Ile Ile Val Asn Asn Ala Arg Gly Ala Ile
Met Xaa Thr 275 280 285 Gln Ala Val Ala Asp Ala Cys Ser Ser Gly Gln
Val Ala Gly Tyr Gly 290 295 300 Gly Asp Val Trp Phe Pro Gln Pro Ala
Pro Lys Gly Pro Pro Trp Arg 305 310 315 320 Tyr Met Pro Asn His Ala
Met Thr Pro His Ile Ser Gly Thr Thr Ile 325 330 335 Asp Ala Gln Leu
Arg Tyr Ala Ala Gly Val Lys Asp Met Leu Asp Arg 340 345 350 Tyr Phe
Lys Gly Glu Asp Phe Pro Val Gln Asn Tyr Ile Val Lys Glu 355 360 365
Gly Gln Leu Ala Ser Gln Tyr Gln 370 375 2 1450 DNA Rice formate
dehydrogenase misc_feature 959, 1296 n = A,T,C or G 2 cgagtcggct
gcactgatcg attccatcac tctctctctc tcgcctgctc gcggttgctg 60
tgcgttcgtc tcgcgatttc tcctcctcct cctgggatca tggcgatgtg gagggcggcg
120 gcggggcatc ttctcggccg cgcgctcggc tccagggccg cgcacacatc
agcaggcagc 180 aagaagatcg tgggtgtgtt ctacaagggc ggcgagtacg
ccgacaagaa tcccaacttc 240 gtcggctgcg tggagggcgc tctcggcatc
cgcgaatggc ttgagtccaa ggggcatcac 300 tacattgtca ccgacgacaa
ggaggggcta aacagcgagc tggagaagca cattgaggat 360 atgcatgtct
tgatcaccac ccctttccac ccagcctatg ttagcgcaga aaggatcaag 420
aaggcaaaga acctcgagct gcttctcaca gctgggattg ggtcagatca tattgatctg
480 ccagcagctg ctgcagcagg tttaactgtg gcagaggtta ccggaagtaa
cactgtgtcg 540 gtggcagaag atgagctcat gcgcattttg attttgctca
ggaacttctt gcccgggtat 600 cagcaggttg ttcatggtga atggaatgtt
gctggcattg cctatagggc ttatgatctt 660 gaaggaaaaa ctgtggggac
tgttggggct ggtcgtattg gcaggctctt acttcagcgt 720 cttaagccct
ttaactgcaa cctgctgtac catgacagac ttaaaattga cccagaactt 780
gagaaagaaa ttggggccaa atatgaagag gatctcgatg ctatgcttcc aaagtgtgat
840 gtcattgtga tcaatacacc tcttacagag aaaacaagag gtatgtttaa
taaagaaaga 900 attgcaaaga tgaagaaagg tgtaatcatt gtgaataatg
ctcgaggagc aatcatggnt 960 acccaggcgg ttgcagacgc ttgctctagt
ggtcaagttg caggctatgg tggtgatgtc 1020 tggttccccc aaccagcacc
aaagggtcca ccctggcggt acatgcctaa tcatgccatg 1080 acccctcata
tctctgggac tacaattgat gcacagctga gatacgcagc aggagttaag 1140
gacatgctgg acaggtactt caaaggggaa gacttcccgg tgcagaacta catcgtcaag
1200 gaaggtcagc tcgcgagcca gtaccagtaa taacccttcc ttgtgtgttg
ggtgtccatc 1260 ctgtggccac cttcccgcag ttaaggggaa cagagnttcg
ggtagggacc aaaacaactt 1320 gtgttgttgt tgttgcctga gtgttccctg
agaaactatt ggacaccgga aataatggat 1380 gcctgccatg gcaaccatgg
ctccgaagaa taaaaagccc tcggattaaa cagtacaaaa 1440 aaaaaaaaaa
1450
* * * * *