U.S. patent application number 12/863986 was filed with the patent office on 2010-12-30 for method for protecting plants from stress and senescence.
Invention is credited to Robert Fluhr, Moshe Sagi.
Application Number | 20100333237 12/863986 |
Document ID | / |
Family ID | 40548603 |
Filed Date | 2010-12-30 |
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United States Patent
Application |
20100333237 |
Kind Code |
A1 |
Fluhr; Robert ; et
al. |
December 30, 2010 |
METHOD FOR PROTECTING PLANTS FROM STRESS AND SENESCENCE
Abstract
The present invention relates to regulation of ureide levels for
optimal plant survival during nutrient remobilization such as
occurs during normal growth, dark stress and senescence. Plants can
be genetically engineered or selected using a suitable gene marker
to have enhanced ureide accumulation. The present invention further
provides methods of protecting plants, or extending the shelf life
of fresh plant produce by application of exogenous ureides.
Inventors: |
Fluhr; Robert; (Rehovot,
IL) ; Sagi; Moshe; (Lehavim, IL) |
Correspondence
Address: |
FENNEMORE CRAIG
3003 NORTH CENTRAL AVENUE, SUITE 2600
PHOENIX
AZ
85012
US
|
Family ID: |
40548603 |
Appl. No.: |
12/863986 |
Filed: |
February 1, 2009 |
PCT Filed: |
February 1, 2009 |
PCT NO: |
PCT/IL2009/000120 |
371 Date: |
September 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61024930 |
Jan 31, 2008 |
|
|
|
Current U.S.
Class: |
800/298 ;
514/263.33; 514/390; 514/563 |
Current CPC
Class: |
A01N 43/50 20130101;
A01N 47/28 20130101; A01N 3/00 20130101; C12N 15/8269 20130101;
A01N 43/90 20130101; C12N 15/8271 20130101; C12N 15/8243
20130101 |
Class at
Publication: |
800/298 ;
514/263.33; 514/390; 514/563 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01N 43/90 20060101 A01N043/90; A01N 43/50 20060101
A01N043/50; A01N 37/30 20060101 A01N037/30; A01P 15/00 20060101
A01P015/00 |
Claims
1. A method for protecting plants or plant parts from a stress
related process comprising applying to the plant or plant part a
composition comprising an amount of at least one ureide effective
to decrease or delay the stress related process.
2. The method according to claim 1 wherein the composition
comprises 0.001-10 mM ureide.
3. The method according to claim 2 wherein the composition
comprises 0.01-10 mM ureide.
4. The method according to claim 2 wherein the composition
comprises 0.1-0.5 mM ureide.
5. The method according to claim 1 wherein the stress related
process is selected from the group consisting of: chlorophyll
degradation, oxidative stress, senescence, premature senescence or
any combination thereof.
6. The method of claim 1 wherein the plant parts are selected from
fruit, vegetables cut branches and flowers.
7. The method of claim 1 wherein the plants are leafy produce.
8. The method of claim 1 wherein the plants or plant parts are
detached from the ground.
9. The method of claim 1 wherein the at least one ureide is
selected from uric acid, allantoin or an allantoate.
10. The method of claim 1 wherein the mode of application is
selected from the group consisting of spraying; at least partially
immersing the plant or plant part in the composition; and adding
the composition to the plant root or plant part medium.
11. (canceled)
12. (canceled)
13. A plant having enhanced ureide production wherein the plant
comprises a genetic variant of at least one enzyme in the pathway
of ureide production from xanthine and wherein the genetic variant
is selected using a probe specific for the at least one enzyme
expression or encoding gene.
14. A plant having enhanced ureide production wherein the plant
comprises a genetic variant of at least one enzyme in the pathway
of ureide utilization and wherein the genetic variant is selected
using a probe specific for the at least one enzyme expression or
encoding gene.
15. A plant having enhanced ureide accumulation wherein the plant
comprises a genetic variant of at least one enzyme in the pathway
of ureide production from xanthine and wherein the genetic variant
is selected using a probe specific for the at least one enzyme
expression or encoding gene.
16. A plant having enhanced ureide accumulation wherein the plant
comprises a genetic variant of at least one enzyme in the pathway
of ureide utilization.
17. (canceled)
18. (canceled)
19. (canceled)
20. A transgenic plant comprising at least one cell transformed
with at least one polynucleotide encoding an enzyme in the pathway
of ureide production from xanthine.
21. The plant of claim 20, wherein said plant produces elevated
concentration of ureide compared to a corresponding non-transgenic
plant.
22. A transgenic plant comprising at least one cell transformed
with at least one polynucleotide encoding an enzyme in the pathway
of ureide utilization.
23. The plant of claim 22, wherein said plant accumulates elevated
concentration of ureide compared to a corresponding non-transgenic
plant.
24. The plant of claim 16 wherein the ureide is selected from
allantoin or an allantoate.
25. The plant of claim 18 wherein the ureide is selected from
allantoin or an allantoate.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of plant survival
and adaptation to environmental stress as well as to the
preservation of fresh plant produce. Particularly, the present
invention relates to the role of ureides in protecting plants
and/or plant produce from senescence or stress related damages.
BACKGROUND OF THE INVENTION
[0002] The remobilization of plant resources during stress
facilitates environmental adaptation. The definitive example of
such metabolite reallocation in a leaf is delineated by leaf
senescence. In addition to natural aging, many environmental
factors such as temperature, drought, nutrient supply, pathogen
attack and light conditions can hasten senescence. For example,
dark-induced stress is characterized by leaf yellowing, due to the
breakdown of chlorophyll and general chloroplast degradation.
Metabolic changes during senescence are associated with the
transition from nutrient assimilation to metabolite turnover that
is accelerated by catabolic activities. For example, in legumes,
the catabolic products of purine, the ureides, can provide the
plant with readily transportable metabolites that excel in a high
nitrogen to carbon ratio. It has been suggested that the carbon
nitrogen ratio generated in energy-conserving purine catabolism may
be critical for the plants survival under stress conditions.
[0003] Little is known about the control of purine catabolism in
leaves. Purine catabolism starts with the conversion of adenosine
monophosphate (AMP) to inosine monophosphate (IMP) by AMP deaminase
(AMPD, E.C.3.5.4.6) that leads by multiple pathways to the
production of oxypurines such as xanthine and hypoxanthine.
However, further degradation of xanthine to urate requires the
activity of the pivotal enzyme, xanthine dehydrogenase (XDH, EC
1.1.1.204). Urate is further metabolized to the ureide, allantoin,
through urate oxidase (UO) and transthyretin-like protein (TLP). In
Arabidopsis, allantoinase (ALN, E.C. 3.5.2.5.) converts allantoin
to allantoate, followed by allantoate amidinohydrolase (AAH, E.C.
3.5.3.9.), that converts allantoate to ureidoglycolate and ammonia.
A putative ureidoglycolate lyase converts ureidoglycolate to the
basic metabolic building blocks, glyoxylate and urea.
[0004] XDH contains molybdenum cofactor (MoCo), FAD and NADPH
binding domains. The second oxygen in the mono-oxo-MoCo is replaced
by a sulfur ligand. When using xanthine/hypoxanthine or NADH as
substrates the sulfo-MoCo form of XDH can generate superoxide
radicals. In contrast, the desulfo-MoCo form of XDH shows only FAD
dependent activity and generats superoxide radical only in the
presence of NADH.
[0005] Among the two XDH encoding genes detected in the Arabidopsis
genome (AtXDH1 and AtXDH2), only AtXDH1 responds to environmental
stimuli. A specific function of XDH in aging is not clear, although
in both mammalian heart and in plants the activity of XDH is
enhanced with age.
[0006] Nowhere in the background art is it taught or suggested that
ureide metabolism can be regulated to increase plant resistance to
stress. Exogenous application of ureides or enhanced endogenous
accumulation of ureides for plant protection and, for example,
extension of shelf life of produce has not been previously known or
suggested in the art.
SUMMARY OF THE INVENTION
[0007] The present invention provides compositions and methods for
protecting plants or plant produce from stress and senescence. The
present invention further provides compositions and methods for
extending the shelf life of plant fresh produce including
vegetables, fruit, cut branches and flowers. In particular, the
present invention provides compositions and methods for protecting
harvested agricultural produce from premature senescence and
senescence.
[0008] It is now disclosed that elevated levels of ureides protect
plants from the deleterious effects of senescence and stress.
Without wishing to be bound by any theory or mechanism of action,
the protection is likely to result from alleviating oxidative
stress by scavenging reactive oxygen species. The present invention
also identifies the enzymes and enzyme variants that regulate the
production of ureides in plants.
[0009] The present invention is based in part on the use of
experimental models of plant senescence and stress such as extended
dark treatment in order to induce metabolite remobilization
processes. It is now disclosed for the first time that both
dark-induced stress and increased age induced accumulation of the
ureides, including allantoin and allantoate, in wild-type leaves.
In contrast, under these conditions, in xanthine dehydrogenase-1
(XDH1) compromised plants, xanthine but no ureides accumulated, and
accelerated senescence and mortality rates were observed. Thus, a
protective role for xanthine dehydrogenase and ureides in
senescence was demonstrated.
[0010] Unexpectedly, this protection can also be achieved by the
exogenous application of ureides to plants or to parts thereof. In
particular, the present invention provides compositions and methods
comprising exogenous ureides for post-harvest protection of plant
fresh produce from senescence and premature senescence.
[0011] According to the principles of the invention it is possible
to utilize any method that increases cellular ureides at critical
times and levels to produce more hardy plants. Plants suffer damage
from oxidative stress during drought, cold and other environmental
abuse. Without wishing to be bound by any theory or mechanism of
action it is envisaged that enhancing the scavenger pathways,
specifically through the presence of higher levels of uriedes,
yields plants that are protected from damage such as damage caused
by environmental stress.
[0012] According to a first aspect, the present invention provides
a method for protecting plants or plant parts from a stress related
process, comprising applying to the plant or plant part a
composition comprising an amount of at least one ureide effective
in decreasing or delaying the stress related process. According to
certain embodiments stress related processes include, among others,
chlorophyll degradation, oxidative stress, senescence, premature
senescence or any combination thereof.
[0013] According to certain embodiments the composition comprises
0.001-10 mM ureide, typically 0.01-10 mM ureide, more typically 0.1
mM-0.5 mM ureide. The composition may comprise 0.1 mM ureide or 0.5
mM ureide or any amount in the above specified ranges.
[0014] According to some embodiments the plant parts are selected
from fruit, vegetables, cut branches and flowers. Other harvested
plant parts may be also be treated. Plants may include, among
others, legumes, herbs and root vegetables or leafy produce (for
example, parsley). As used herein, the term "harvest" refers to the
gathering of agricultural plant products, including picking (e.g.
fruit), detaching from the ground (e.g. leafy crops such as
lettuce, spinach and parsley), cutting (e.g. branches, including
ornamental branches) and the like.
[0015] According to certain embodiments the ureide is selected from
allantoin or allantoate.
[0016] According to some embodiments applying the composition to
plants includes spraying the plants or plant parts with the
composition. According to other embodiments applying includes
adding the composition to the plant's root or plant's branch
medium. According to further embodiments, the composition is
applied by at least partially immersing the plant or plant part in
the composition. A combination of applying methods may also be used
according to embodiments of the invention.
[0017] Elevated ureide amounts within a plant cell may be achieved
by selecting a variant plant or genetically engineering the plant
to produce or accumulate higher ureide concentration as compared to
a wild type plant. The selected or engineered plants can
overexpress enzymes involved in the production pathway of ureides,
or have decreased expression levels of ureide utilization enzymes
such that ureide is accumulated. The selection for the plant may
include using a probe specific for at least one enzyme related to
the production or accumulation of ureides and/or selecting a mutant
having enhanced purine turnover.
[0018] Thus, according to another aspect the present invention
provides a plant having enhanced ureide production wherein the
plant comprises a genetic variant of at least one enzyme in the
pathway of ureide production from a xanthine.
[0019] According to another aspect, the present invention provides
a plant having enhanced ureide accumulation wherein the plant
comprises a genetic variant of at least one enzyme in the pathway
of ureide utilization.
[0020] According to a further aspect the present invention provides
a transgenic plant comprising at least one cell transformed with at
least one polynucleotide encoding an enzyme in the pathway of
ureide production from a xanthine. According to certain
embodiments, the transgenic plant produces elevated concentration
of ureide compared to a corresponding non-transgenic plant.
[0021] According to yet additional aspect the present invention
provides a transgenic plant comprising at least one cell
transformed with at least one polynucleotide encoding an enzyme in
the pathway of ureide utilization. According to certain
embodiments, the transgenic plant accumulates elevated
concentration of ureide compared to a corresponding non-transgenic
plant.
[0022] These and other embodiments of the present invention will
become apparent in conjunction with the figures, description and
claims that follow.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 demonstrates XDH expression in Arabidopsis wild type
and Atxdh1 modified plants. FIG. 1a shows Atxdh1 and Atxdh2
relative expression monitored by quantitative real-time RT-PCR
analysis using wild-type (Col, left panel) and XDH compromised
plants (KO, SALK.sub.--148364 Atxdh1 T-DNA insertion line; and Ri,
three xdh RNA interference lines, right panel). FIG. 1b shows
XDH-dependent NADH oxidase and FIG. 1c shows XDH-dependent
hypoxanthine/xanthine dehydrogenase activities in wild type (Col,
left panel) and KO (XDH compromised plants, right panel) leaf
extracts. The O.sub.2.sup.- generated in-gel was measured using NBT
as electron acceptor and presented relative to day 0 or, during the
recovery period, relative to day 6 in the dark. Each lane contained
200 .mu.g of soluble proteins. FIG. 1d demonstrates O.sub.2.sup.-
generating activity of XDH in leaf extracts of wild-type and XDH
compromised plants. Plants were treated and sampled as described
above. Kinetics was assayed using epinephrine as electron acceptor
and hypoxanthine/xanthine as substrate following by adenochrome
absorbance. Values are means.+-.SE (n=6).
[0024] FIG. 2 demonstrates the responses of wild-type and XDH
compromised Arabidopsis plants to dark stress. FIG. 2a shows
wild-type (Col) and XDH compromised plants (KO, SALK.sub.--148364;
Ri, xdh1 RNA interference) during 6 day dark treatment and
subsequent 9 days recovery. FIG. 2b shows survival rates of
wild-type and XDH compromised plants 15 days after exposure to the
conditions described in FIG. 2a. Error bars indicate SE (n=12).
FIG. 2c shows leaves of control untreated and dark-treated plants
and XDH compromised plants excised before (0) and 6 days after (6)
exposure to dark. FIG. 2d demonstrates the damage level of leaves
shown in FIG. 2c calculated as described in the experimental
procedures. Bars show means.+-.SEM (n=6). FIG. 2e shows chlorophyll
content in leaves sampled over a 6 day dark treatment. Error bars
indicate SE (n=6). FIG. 2f shows total soluble protein content in
leaves sampled over a 6 day dark treatment. Error bars indicate SE
(n=6). FIG. 2g demonstrates the progress in cell death over a 4 day
dark treatment as indicated by staining with trypan-blue. At least
8 leaves were examined for each condition, and representative
fields having the same magnification are shown.
[0025] FIG. 3 demonstrates relative transcript expression of
selected senescence and chlorophyll degradation-related genes.
Transcripts were monitored by quantitative real-time RT-PCR
analysis using wild-type (Col) and XDH compromised plants (KO,
SALK.sub.--148364, Atxdh1 T-DNA insertion line and Ri, mean of 3
independent xdh1 RNA interference lines). Plants were sampled
before being placed in the dark (0) and over a dark period of six
days. The transcript expressions of each treated line was compared
to the untreated line after normalization to the Arabidopsis
EF-1.alpha. gene product (At5g60390) and presented as relative
expression. Values are means.+-.SEM (n=6).
[0026] FIG. 4 shows Nitroblue tetrazolium (NBT)-O.sub.2.sup.-
generation in wild type (Col) and xdh1 RNA interference (Ri) plants
in response to dark treatment. FIG. 4a demonstrates superoxide
level as visualized by NBT staining of rosette leaves in the
absence or presence of allopurinol (+Allo) or diphenylene iodonium
(+DPI). FIG. 4b shows quantitative analysis of NBT-O.sub.2.sup.-
production in leaves in response to dark treatment.
NBT-O.sub.2.sup.- generation was quantified in stained leaves by
scanning. Upper insert: total relative NBT-O.sub.2.sup.+
generation; middle insert: relative NBT-O.sub.2.sup.+ generation by
XDH obtained by subtraction of +Allo from -Allo leaf scan values;
lower insert: relative NBT-O.sub.2.sup.+ generation by non-XDH
source, obtained by subtraction of +DPI from +Allo leaf scan
values. Data are means.+-.SE (n=8).
[0027] FIG. 5 shows analysis of the purine metabolites, xanthine,
allantoin and allantoate, in response to dark stress. Xanthine
(FIG. 5a), Allantoin (FIG. 5b) and Allantoate (FIG. 5c) were
determined in rosette leaves of wild-type (Col) and XDH1
compromised plants (KO, SALK.sub.--148364; Ri, xdh1 RNA
interference) after being kept in dark for 6 days and transferred
to 16 h light/8 h dark regime for recovery during additional 9
days. Values are means.+-.SEM (n=6).
[0028] FIG. 6 demonstrates transcript expression levels of
Arabidopsis purine catabolism genes, AMP deaminase (AMPD),
allantoinase (ALN) and allantoate amidinohydrolase (AAH) during
dark stress and subsequent recovery period. Quantitative analysis
of transcripts by real-time RT-PCR was performed using wild-type
(Col) and XDH compromised plants (KO, SALK.sub.--148364 Atxdh1
T-DNA insertion line; Ri, means of three xdh RNA interference
lines) kept in the dark for 6 days and transferred to a 16 h
light/8 h dark regime for recovery during additional 9 days. The
expressions of each dark treated line was compared to the untreated
line after normalization to the Arabidopsis EF-1.alpha. gene
product (At5g60390) and is presented as the relative expression on
day 0 or, for the recovery period, compared to the value of the
dark treated line after 6 days in dark. Values are means.+-.SEM
(n=3).
[0029] FIG. 7 demonstrates the influence of aging on leaf
phenotype, purine metabolite level and transcript expression in
wild type and XDH1 compromised plants. FIG. 7a shows 75 days old
wild type (Col) and xdh1 RNA interference (Ri) plants and their
representative leaves (left and middle panels). The transcript
expression of SAG12 measured by quantitative real-time RT-PCR and
normalized to the EF-1.alpha. transcript (At5g60390) is shown at
the right panel. Values are means.+-.SEM (n=6). Data for Ri lines
are means of 3 independent lines. FIG. 7b shows concentration of
the purine metabolites xanthine (left panel), allantoin (middle
panel) and allantoate (right panel), measured in rosette leaves, in
response to plant age. Values are means.+-.SEM (n=6). FIG. 7c
demonstrates transcript expression of Arabidopsis purine catabolism
genes using wild-type (Col) and three independent Ri plants. The
transcript expressions are relative to the level at 30 days and
normalized to the EF-1.alpha. transcript (At5g60390). Values are
means.+-.SEM (n=3).
[0030] FIG. 8 demonstrates the responses of Arabidopsis wild-type
(Col) and xdh RNA interference (Ri) plants to application of the
purine catabolites: xanthine, allantoin and allantoate. FIG. 8a:
xanthine (1 mM) in the presence or absence of allopurinol (1 mM,
+/-Allo), was applied for 48 h to leaf discs placed in the dark and
photographed 24 h after being transferred to 16 h light/8 h dark
regime (left upper panel). Remaining chlorophyll, xanthine,
allantoin and allantoate were determined in leaf discs sampled at
the beginning of the experiment (light) or after 72 h (as described
above) without xanthine (dark). FIG. 8b: allantoin or allantoate
(0.1 mM) in the presence or absence of allopurinol (1 mm, +/-Allo)
was applied to leaf discs for 2 h in the dark after keeping the
discs in the dark for 48 h. Discs were photographed 24 h after
being transferred to 16 h light/8 h dark regime (left upper panel).
Leaf discs were washed twice before sampling for xanthine,
allantoin and allantoate determination (right and bottom panels).
Bars show mean.+-.SEM (n=16).
[0031] FIG. 9 shows reactive oxygen species (ROS) accumulation in
the presence of allantoin and allantoate in Arabidopsis wild-type
(Col) and xdh1 RNA interference (Ri) lines. FIG. 9a left panel::
leaf discs removed from Col plants placed for 24 h in the dark in
the presence or absence of allantoin or allantoate (0.1 mM) and
then washed and treated with H.sub.2O.sub.2 (0, 20, 50 mM) for an
additional 6 h in the dark. Right panel: remaining chlorophyll
determined 24 h after the disc were transferred to a 16 h light/8 h
dark regime. Bars show mean.+-.SEM (n=16). FIG. 9b shows
H.sub.2O.sub.2 production in leaf discs of Col and Ri plants
treated with allantoin or allantoate (0.1 mM) visualized by
staining with 3,3'-diaminobenzidine (DAB) (left panel). Total
H.sub.2O.sub.2 relative production (right insert) was quantified in
leave discs kept in the dark for 24 h and then treated with
allantoin or allantoate for additional 2 h in the dark, using DAB
staining Results are from two independent experiments. Error bars
indicate SE (n=16). The lower case letters (a, b) indicate
P<0.001 for the differences within treatment between ecotypes.
Capital letters (A, B) indicate P<0.001 for the differences
within ecotypes between treatments. FIG. 9c demonstrates the
influence of allantoin or allantoate (0.1 mM) on O.sub.2 production
in leaf discs of Col and Ri plants. Leaf discs were treated and
analyzed as in FIG. 9b above. Total relative superoxide production
was quantified in leaf discs from 2 independent experiments. Error
bars indicate SE (n=16).
[0032] FIG. 10 shows transcript expression of the Arabidopsis Cys
protease senescence-associated gene 12 (SAG12), during 6 days of
dark stress. Quantitative analysis of transcript by real-time
RT-PCR was performed using wild-type (Col) and three independent
RNA interference (Ri) lines. Plants were sampled before dark
application (0) and over the six days dark period. The expressions
of each treated line were compared to those of the untreated line
after normalization to the Arabidopsis EF-1.alpha. gene product
(At5g60390) and data are presented as relative expression. Values
are means.+-.SEM (n=6). Data for Ri lines are means of 3
independent lines.
[0033] FIG. 11 shows transcript expression of the Arabidopsis
molybdenum cofactor sulfurase gene, ABA3, in wild type (Col) plants
after exposure to dark stress for six days. Transcript Analysis was
performed as described in FIG. 10 above.
[0034] FIG. 12 shows transcript expression of Arabidopsis purine
catabolism genes, encoding urate oxidase (UO) and
transthyretin-like protein (TLP), is wild type (Col) plants exposed
to dark stress of 6 days and subsequent recovery period of 16 h
light/8 h dark regime for 9 days. Quantitative analysis of
transcripts was performed by real-time RT-PCR compared to the
untreated plant after normalization to the Arabidopsis EF-1.alpha.
gene product (At5g60390). Data are presented as relative expression
compared to day 0 or, for the recovery period, compared to the
value of the dark treated plant after 6 days in the dark. Values
are means.+-.SEM (n=3).
[0035] FIG. 13 shows diurnal fluctuation of allantoin in rosette
leaves of wild-type (Col) Arabidopsis plants. Values are
means.+-.SEM (n=6).
[0036] FIG. 14 relates to the influence of allantoin application on
senescence in parsley leaf discs exposed to dark stress. Parsley
leaf discs were kept for 2 days in the light and then transferred
to the dark for additional 2 days with or without the addition of 0
to 10 mM allantoin. CL-control undetached leaves in the light.
[0037] FIG. 15 demonstrates the response of tomato to extended dark
stress and recovery in light thereafter. FIG. 15a shows the
allantoin level in the 1st, 3rd and 5th leaf of wild type tomato
plants over a period of 12 days in the dark and 10 days of recovery
in the light. FIG. 15b shows that application of 0.1 mM and 0.5 mM
allantoin delays chlorophyll degradation in tomato leave discs
(treated as in FIG. 14).
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides compositions and methods for
protecting plants and parts thereof from senescence and stress
related processes. The present invention further provides
compositions and methods for extending the shelf life of plant
fresh produce including, but not limited to, herbs, vegetables, cut
branches, fruit and flowers. Without wishing to be bound by any
theory or mechanism of action, shelf life extension of the fresh
produce according to the teachings of the present invention may be
attributed to protection of the harvested material from processes
such as senescence, premature senescence and stress related
processes.
[0039] According to certain embodiments, the present invention
relates to regulation of ureide levels for optimal plant survival
during nutrient remobilization such as occurs during normal growth,
dark stress, senescence and other plant processes.
[0040] According to some embodiments, plants can be genetically
engineered or selected using a suitable gene marker to achieve
enhanced ureide production and/or accumulation.
[0041] According to some embodiments the present invention
identifies enzymes and enzyme variants that regulate the production
of ureides in plants.
[0042] The present invention is based in part on the use of
experimental models of plant senescence and stress such as extended
dark treatment in order to induce metabolite remobilization
processes. It is now disclosed for the first time that both
dark-induced stress and increased age induce accumulation of
ureides, including allantoin and allantoate, in wild-type leaves.
In contrast under these conditions, in XDH1-compromised plants,
xanthine but no ureides accumulate, and accelerated senescence and
mortality rates are observed. Thus, a protective role for AtXDH1
and ureides in senescence is demonstrated.
[0043] Unexpectedly, this protection can also be achieved by the
exogenous application of ureides. In particular, the present
invention provides compositions and methods comprising exogenous
ureides for protection of picked or harvested agricultural produce
from processes, such as senescence and premature senescence.
[0044] According to a first aspect, the present invention provides
a method for protecting plants, plant parts or harvested plant
produce from aging (for example, senescence or premature
senescence). According to one embodiment, the method comprises
applying to the plant(s) or plant part(s) a composition comprising
an amount of at least one ureide effective to decrease or delay the
senescence of the plant or plant parts. According to some
embodiments the ureide is selected form allantoin or an
allantoate.
[0045] According to one embodiment plants or plant parts may be
selected from harvested herbs, fruits, vegetables and flowers.
[0046] In particular, the present invention provides compositions
and methods for protecting harvested agricultural produce from
typically stress related processes such as senescence and premature
senescence. Picked flowers and leafy crops such as lettuce,
spinach, parsley and many others typically begin to undergo
senescence immediately after they are picked. This process is
typically accelerated during storage of the plant produce.
Typically, this means that the green leafy tissue yellows and its
nutritional and marketing value may decrease. Embodiments of the
present invention thus disclose that the exogenous application of
ureides slows this progress and extends plant produce shelf
life.
[0047] Methods, according to some embodiments of the invention, may
comprise applying, such as by applying to the root or cut branches
medium or spraying, a composition which includes an appropriate
amount of ureides onto the produce or immersing or partially
immersing the produce in a suspension or other appropriate form of
composition containing ureides or purine metabolites such as uric
acid. Any suitable application method, such as, but not limited to,
the methods listed here, may be used alone or in combination.
According to some embodiments any solution comprising ureides in
the range of 1 .mu.M-10 mM may be used. Typically a solution in a
range of 0.01-10 mM, or 0.1-10 mM is used. Any concentration of
ureides in the specified ranges may be used. According to one
embodiment 0.1 mM ureide, 0.5 mM ureide or any concentration of
ureides in this range may be used. Compositions having other
concentrations may also be used with embodiments of the
invention.
[0048] As exemplified hereinbelow application of exogenous ureides
protects plants and plant parts from senescence and stress related
processes.
[0049] According to another aspect of the invention ureide
accumulation may be enhanced in genetically engineered plants. In
the alternative, plants may be genetically engineered to achieve
enhanced and optimal ureide production. Expression of enzymes
specifically required in the pathway of ureide production may be
optimized, according to one embodiment of the invention, in order
to achieve ureide accumulation. According to further aspects the
present invention provides a plant selected for enhanced ureide
production. According to one aspect a genetically modified plant
can be obtained by enhancing expression levels of enzymes involved
in the production pathway of ureides thereby increasing ureide
production. According to another aspect a genetically modified
plant can be obtained by decreasing expression levels of ureide
utilization enzymes by genetic engineering thereby increasing
ureide accumulation.
[0050] According to another aspect the desired plant can be
selected using a probe specific for at least one enzyme related to
the production or accumulation of ureides. According to one
embodiment the desired plant can be selected using a probe specific
for XDH encoding gene or XDH enzyme expression.
[0051] According to one embodiment a genetically modified plant is
selected for mutations for enhanced purine turnover. A specific
controlling element may up and down regulate purine catabolic
pathways synchronously, i.e. up regulate the enzymes that make
ureides and simultaneously down-regulate the enzymes that can
breakdown the ureides. Such selection can be done by various known
mutational methods e.g., subjecting plants to mutagenesis as in
enhancer trap methods, irradiation, chemical mutagenesis,
insertional mutagenesis and other appropriate methods. According to
one embodiment an enhancer is inserted randomly in the genome and
the resultant lines are screened for changes in ureide levels.
According to various embodiments generic lines may be used for
screening of ureide production.
[0052] According to another aspect identification of transcripts
relevant to the ureide metabolism (production or degradation) can
be used to perform large scale screening for natural variations in
ureide levels in plants in response to stress. According to a
particular embodiment the natural variants in ureide levels are
screened during or after extended dark periods.
[0053] It should be understood that composition and/or methods
according to embodiments of the invention may be used alone or in
combination to achieve results according to the invention. For
example, several different concentrations and/or different ureides
may be applied to plant, plant parts or crops, according to
embodiments of the invention. Genetic engineering and/or screening
methods may be used in combination with methods of applying ureides
to plants. Any combination of the methods and/or compounds
disclosed herein, which achieves protection of plants through the
ureide metabolic pathway, is disclosed herein.
[0054] The remobilization of metabolites during stress and
senescence plays an important role in optimal plant adaptation to
the environment. The plant molybdenum-cofactor (MoCo) and flavin
containing enzyme, xanthine dehydrogenase (XDH; EC 1.2.1.37) are
pivotal for purine remobilization and catalyze the conversion of
the purine catabolic products, hypoxanthine and xanthine to uric
acid that is subsequently degraded to the ureides, allantoin and
allantoate. The present invention now shows that in wild-type
plants, conditions of extended darkness or increasing leaf age
cause induction of transcripts related to purine catabolism, that
in turn result in marked accumulation of the purine catabolic
products, allantoin and allantoate. In contrast, Arabidopsis
mutants of XDH, Atxdh1, accumulated xanthine and showed premature
senescence symptoms as exemplified by enhanced chlorophyll
degradation, extensive cell death and up-regulation of
senescence-related transcripts. When dark-treated mutant lines were
re-exposed to light, they showed elevated levels of reactive oxygen
species (ROS) and higher mortality rate compared to wild-type
plants. Unexpectedly, the level of ROS and mortality could be
attenuated by the addition of allantoin and allantoate suggesting
that these metabolites can act as scavengers of reactive oxygen
species. The present invention highlights a yet unrecognized need
for the controlled maintenance of ureide levels, for example, as
mediated by AtXDH1 activity during dark stress and aging and point
to the dual functionality of ureides as efficient stores of
nitrogen and as cellular protectants. Thus, regulation of ureide
levels by regulating Atxdh1 expression has general implications for
optimal plant survival during nutrient remobilization such as
occurs during normal growth, dark stress and senescence.
XDH1 Activity Moderates Pre-Mature Senescence Induced by Dark
Stress
[0055] Extended dark treatment implies complete lack of new
photosynthesis that imposes a stress which requires remobilization
of leaf resources. In this sense, extended dark initiates molecular
events that are reminiscent of leaf senescence. The progression of
senescence must be orderly so that the accumulation of deleterious
compounds or physiological states of enhanced labile cellular state
is avoided. It is shown here that XDH1 activity is required for
normal remobilization to succeed, as in its absence, not only will
direct purine catabolism be compromised, but the rate of
chlorophyll, soluble protein degradation (FIGS. 2e and 2f) and cell
death are all accelerated.
[0056] The value of extended dark treatments as a signal that
stimulates synchronized metabolic remobilization in the plant was
discerned by examining transcript induction profiles of genes that
are known to be induced during senescence. For example, the WRKY53
transcription factor (which was shown to control early events of
senescence and oxidative stress response (Miao Y. et al., 2004
Plant Mol Biol 55, 853-867). The protease regulator, ERD1 encodes
caseinolytic protease regulatory subunit, and may be related to
posttranslational modification processes, as it was shown to be
involved in the degradation of chloroplast proteins subjected to
artificial senescence related to ABA, ethylene and SO.sub.2
(Brychkova G. et al., 2007 Plant J 50, 696-709; Weaver L. M. et
al., 1998 Plant Mol Biol 37, 455-469). Similarly, chlorophyll
degradation (Gepstein S. 2004 Genome Biology 5, 212; Pruzinska A.
et al., 2005 Plant Physiol 139, 52-63) and up-regulation of related
transcripts such as ACD1 and SGN1 (Park S. Y. et al., 2007 Plant
Cell 19, 1649-1664; Tanaka R. et al., 2003 Plant Cell Physiol 44,
1266-1274), during the extended dark period are indicative of leaf
senescence. The up-regulation of the chlorophyll-degradation
related transcripts and chlorophyll degradation itself is a
hallmark of compromised XDH1 activity (FIG. 2e and FIG. 3, lower
panels). Thus, as indicated by these marker genes, the presence of
XDH1 activity affords wild-type plants protection from the onset of
program cell death-like process during the dark stress.
XDH Activity in Response to Dark Stress
[0057] XDH possesses a MoCo sulfurated-dependent
hypoxanthine/xanthine-O2-generating activity and a FAD dependent
NADH-OT-generated activity (Sagi M. et al., 1999 Plant Physiol 120,
571-578; Sagi M. et al., 2002 Plant J 31, 305-317; Yesbergenova Z.
et al., 2005 Plant J 42, 862-876). The xanthine-dependent
superoxide generating activity was more enhanced in extended dark
than the NADH-dependent activity (FIGS. 1b-1d and FIG. 4b middle
panel). This indicates that modification processes involving the
MoCo-domain are likely taking place during the dark-derived
remobilization. Difference between MoCo and FAD dependent
activities of XDH may result from interconversion of the sulfo- and
desulfo-MoCo forms of XDH. The terminal sulfuration step carried
out by the MoCo-sulfurase enzymes FLACCA in tomato and ABA3 in
Arabidopsis were hypothesized to provide an efficient way to
regulate XDH activities by sulfuration (Sagi et al., 1999, ibid;
Sagi et al., 2002 ibid). XDH commonly contains significant portions
of desulfo-forms, which while inactive in hypoxanthine/xanthine
oxidases (Harrison R. 2002 Free Radic Biol Med 33, 774-797), are
active as NADH oxidases (Yesbergenova et al., 2005, ibid). Thus,
the enhancement in hypoxanthine/xanthine oxidase activity observed
in dark-induced stress may result from enhanced sulfuration status
of the XDH MoCo domain. Consistent with this scenario, it is noted
that the ABA3 transcript was significantly enhanced during dark
stress (about 3-fold; FIG. 11).
Role of Purine Catabolism in Dark Stress and Recovery
[0058] Mutation in AtXDH1 or application of allopurinol to wild
type plants led to xanthine accumulation during dark stress (FIGS.
5a and 8a, 8b). The degree of tissue damage that appears in plants
during the dark and especially during the subsequent light
treatment may be due to direct or indirect sensitivity to excess
xanthine, leading to chlorophyll degradation, especially in the
absence of active AtXDH1 in the treated leaves (FIG. 8a). However,
the higher chlorophyll degradation that appeared in mutant leaves
containing high level of xanthine was significantly alleviated by
the addition of allantoin and allantoate implying that ureides
protected leaves from dark induced chlorophyll degradation in spite
of xanthine accumulation (FIG. 8b). Thus, ureides accumulation may
contribute to the cell normal growth pattern through a critical
physiological state brought upon by metabolite remobilization.
Hence, ureides represent both favorable N--C ratio as well as
molecules with an intrinsic ability to scavenge ROS excess. Such
excess may be encountered during re-exposure of leaves to light in
which photosystem imbalance may be the end result of enhanced
protein turnover that results in transient light-dependent ROS
production.
[0059] A coordinated rise in transcripts level involved in ureide
production (AMPD, XDH1, UO and TLP) and a concomitant decrease in
transcripts level of enzymes that utilize ureides (ALN and AAH) was
detected. Thus, dark stress ureides accumulation is at least in
part under transcriptional control and is likely to result from the
difference in activities involved in ureides formation versus the
activities that process them.
[0060] The data presented in the present invention have highlighted
a complete and functional purine remobilization pathway that occurs
in the extended absence of light but the implications are not
restricted to conditions of environmental stress. Rapid XDH1
up-regulation (FIGS. 1a and 1c) and a mild increase in ureide
levels in normal diurnal light dark transitions (22%; FIG. 13) are
noted. The diurnal cycling of ureide levels indicates that extended
dark treatment accentuates a normal response. In this respect, the
physiological and molecular components of dark-induced stress are
shown here to be reminiscent of normal leaf aging and senescence.
Indeed in dark stress and senescence similar levels of ureides
accumulation was shown (FIGS. 5 and 7).
[0061] Senescence and dark-induced senescence are associated with
modulation of ROS production (Guo F. Q. and Crawford N.M. 2005
Plant Cell 17, 3436-3450; Pastori G. M. and Del Rio L. A. 1997
Plant Physiol 113, 411-418). Thus, without wishing to be bound by
any particular theory opr mechanism of action, ureides may serve a
dual function in senescence as transportable metabolites for
further biosynthetic pathways and at the same time as cellular
protectants from ROS. The results herein show that purine
catabolism, aging and extended dark induce similar senescence
programs. In both cases, the transition from nutrient assimilation
to metabolite turnover occurs due to the acceleration of purine
catabolic recycling activities in which AtXDH1 plays a pivotal
role.
[0062] Examples of specific experiments carried out on specific
plants are presented herein in order to more fully illustrate
certain embodiments of the invention. They should in no way,
however, be construed as limiting the broad scope of the invention.
One skilled in the art can readily devise many variations and
modifications of the principles disclosed herein without departing
from the scope of the invention.
EXAMPLES
Experimental Procedures
Plant Materials, Growth Conditions and Dark Treatment
[0063] Arabidopsis thaliana (ecotype Columbia) and its isogenic
Atxdh1 compromised plants were grown in a growth room under 16 h
light/8 h dark, 22.degree. C., 75-85% relative humidity, light
intensity of 100 .mu.mol m.sup.-2 s.sup.-1 as described before
(Brychkova G. et al., 2007, ibid). Atxdh1 compromised plants
included homozygous T-DNA inserted line [SALK.sub.--148364
(obtained from the Arabidopsis Biological Research Center,
Colombus, Ohio, USA)] and 3 independent homozygous xdh RNA
interference lines described before (Yesbergenova et al., 2005,
ibid). For dark treatment, four-week-old plants grown in peat were
transferred from the growth room to a dark room. Samples were
collected every day during 15 min, under dim light (40 .mu.mol m-2
s-1), as a mix of fully expanded rosette leaves taken from 5
plants. After 6 days in the dark, plants were transferred to the
growth room and survival rate of plants were determined 9 days
later. Average and S.E. of survival rate was calculated from 12
independent experiments with at least 30 plants for each treatment.
For aging measurements plants were grown in a growth room as
described above and samples were collected from 30, 45, 60 and 75
day old plants as a mix of fully expanded rosette leaves taken from
5 plants.
Cell Death Measurements, Determination of Chlorophyll and Leaf
Damage Level
[0064] Cell death was visualized in leaves sampled from dark
stressed and control unstressed plants, by lactophenol-trypan blue
staining followed by destaining in saturated chloral hydrate (Koch
E. and Slusarenko A. 1990 Plant Cell 2, 437-445). Total chlorophyll
content was measured in extracts of the fully expanded leaves as
described before (Brychkova et al., 2007, ibid). The values for
remaining chlorophyll content in leaf discs were determined as the
amount of chlorophyll per disc divided by the amount of chlorophyll
per untreated control and expressed as a percentage. The severity
of leaf damage after dark stress was as follows: 1, no damage; 2,
<30%; 3, 30-50%; 4, >50% of the leaf area damaged. The
average leaf damage was then multiplied by the total number of
damaged leaves to determine the damage level. Means.+-.SEM for each
treatment are presented.
Preparation of RNA and Quantitative Real-Time RT-PCR
[0065] For quantitative analysis of transcripts expression, total
RNA, RT reaction and quantitative RT-PCR reactions with specific
primers (see Table 1 hereinbelow) were performed as describes in
Brychkova et al. (2007, ibid). Reactions normalized with UBQ10
(At4g05320) and Elongation factor 1-.alpha. (At5g60390) as
housekeeping genes revealed similar results and thus only results
based on the later housekeeping gene are presented.
TABLE-US-00001 TABLE 1 List of primers used for quantitative
real-time PCR with Arabidopsis thaliana Length of PCR Tran- PCR
product Primer script SGN1 Forward 189 (StayGreen1;
GATTGTTCCCGTTGCAAGGTTGTTT At4g22920) (SEQ ID NO: 1) Reverse
TGCAACTGAGAGTTGTTTATGGATTGAG (SEQ ID NO: 2) SRG1 forward 191
(senescence- AAGAGTGGGGATTTTTCCAGCTTGT related gene 1; (SEQ ID NO:
3) At1g17020) reverse TGCCCAATCTAGTTTCTGATCTTCTGA (SEQ ID NO: 4)
UBQ10 forward 192 (Ubiquitine10; TTTGTTAAGACTCTCACCGGAAAGACA
At4g05320) (SEQ ID NO: 5) reverse GAGGGTGGATTCCTTCTGGATATTGTA (SEQ
ID NO: 6) UO (Urate forward 187 oxidase;
CACTGTTTATGTGAAAGCCAAGGAATG At2g26230) (SEQ ID NO: 7) reverse
CCCAAGCTTAAAACCATGTAAATGTGG (SEQ ID NO: 8) TLP forward 185
(transthyretin- CCATGCGTTAAAGGAAAGGTATGAAAA like protein; (SEQ ID
NO: 9) AT5g58220) reverse TGATTCTCAGACGATCTTGAGGTTTTG (SEQ ID NO:
10) WRKY53 forward 158 (WRKY DNA- TCAAAGAAAAGAAAGATGTTACCAAAGTGG
binding protein (SEQ ID NO: 11) 53; At4g23810), reverse
GTGCATCTGTAATAACTCCTTGGGAAT (SEQ ID NO: 12) XDH1 (xanthine forward
200 dehydrogenase TGATGTTGGACAAATAGAAGGAGCGTTT 1; At4g34890) (SEQ
ID NO: 13) reverse TATTCGGATTCCCCTTGAGAAGCGAAACA (SEQ ID NO: 14)
XDH2 (xanthine Forward 202 dehydrogenase
TGATATTGGACAAATAGAAGGAGCGTTT 2; At4g34900) (SEQ ID NO: 15) reverse
TGCATTTGGATTACCCTTGAGAAGAGAAA (SEQ ID NO: 16) XERO1/TAS14 forward
165 (dehydrin; AGACTCACCAACAGCTTGACCAATTT At3g50980) (SEQ ID NO:
17) reverse CACCTAGTCCATCATCCGAGCTAGAG (SEQ ID NO: 18) SGN1 Forward
189 (StayGreen1; GATTGTTCCCGTTGCAAGGTTGTTT At4g22920) (SEQ ID NO:
19) Reverse TGCAACTGAGAGTTGTTTATGGATTGAG (SEQ ID NO: 20) SRG1
forward 191 (senescence- AAGAGTGGGGATTTTTCCAGCTTGT related gene 1;
(SEQ ID NO: 21) At1g17020) reverse TGCCCAATCTAGTTTCTGATCTTCTGA (SEQ
ID NO: 22) UBQ10 forward 192 (Ubiquitine10;
TTTGTTAAGACTCTCACCGGAAAGACA At4g05320) (SEQ ID NO: 23) reverse
GAGGGTGGATTCCTTCTGGATATTGTA (SEQ ID NO: 24) UO (Urate forward 187
oxidase; CACTGTTTATGTGAAAGCCAAGGAATG At2g26230) (SEQ ID NO: 25)
reverse CCCAAGCTTAAAACCATGTAAATGTGG (SEQ ID NO: 26) TLP forward 185
(transthyretin- CCATGCGTTAAAGGAAAGGTATGAAAA like protein; (SEQ ID
NO: 27) AT5g58220) reverse TGATTCTCAGACGATCTTGAGGTTTTG (SEQ ID NO:
28) WRKY53 (WRKY forward 158 DNA-binding
TCAAAGAAAAGAAAGATGTTACCAAAGTGG protein 53; (SEQ ID NO: 29)
At4g23810), reverse GTGCATCTGTAATAACTCCTTGGGAAT (SEQ ID NO: 30)
XDH1 (xanthine forward 200 dehydrogenase
TGATGTTGGACAAATAGAAGGAGCGTTT 1; At4g34890) (SEQ ID NO: 31) reverse
TATTCGGATTCCCCTTGAGAAGCGAAACA (SEQ ID NO: 32) XDH2 (xanthine
Forward 202 dehydrogenase TGATATTGGACAAATAGAAGGAGCGTTT 2;
At4g34900) (SEQ ID NO: 33) reverse TGCATTTGGATTACCCTTGAGAAGAGAAA
(SEQ ID NO: 34) XERO1/TAS14 forward 165 (dehydrin;
AGACTCACCAACAGCTTGACCAATTT At3g50980) (SEQ ID NO: 35) reverse
CACCTAGTCCATCATCCGAGCTAGAG (SEQ ID NO: 36)
Histochemical Staining for O.sub.2.sup.- and H.sub.2O.sub.2
Detection
[0066] Nitroblue tetrazolium (NBT) O.sub.2.sup.- staining of leaves
was performed essentially according to Jabs T. et al. (1996 Science
273, 1853-1856) and Fryer M. J. et al. (2002 J Exp Bot 53,
1249-1254). Leaves were incubated for 10 h in the dark overnight
and then placed in 96% ethanol, boiled for 10 min and photographed.
Application of allantoin and allantoate and O.sub.2'' and
H.sub.2O.sub.2 measurement was carried out on 7 mm leaf discs
removed from 4-week-old Col wild-type and Atxdh1 mutant plants.
Discs were placed in the dark for 24 h on moistened filter papers.
Thereafter, 0.1 mM allantoin or allantoate in pH 7.5 solution was
added to the filter papers for additional 2 h. For
NBT-O.sub.2.sup.- staining, leaf discs were washed twice and
immersed in 0.8 mM NBT solution added to the filter papers for 2 h
in the light and then boiled in 96% ethanol for 10 min and
photographed. For DAB-H.sub.2O.sub.2 staining leaf discs subjected
to the procedure described above for NBT were immersed in solution
containing 1 mg ml.sup.-1 3,3'-diaminobenzidine (DAB) in pH 5.0,
for 2 h in the light as described before (Sagi M. et al., 2004
Plant Cell 16, 616-628). Leaf discs were photographed after boiling
in 96% ethanol for 10 min. NBT and DAB stained leaves and leaf
discs were quantified using NIH Image Software (Version 1.6).
Protein Extraction, Fractionation and Total Soluble Protein
Determination in-Gel and Kinetic Assay of ROS-Generating Activities
of XDH
[0067] Extracts of XDH for assay by native gel electrophoresis
(PAGE) were prepared as described by Sagi M. et al. (1998 Plant
Science 135, 125-135) and Yesbergenova et al. (2005, ibid). Kinetic
O.sub.2.sup.- generating activities of XDH were assayed
spectrophotometrically by measuring the oxidation of epinephrine to
adrenochrome at 480 nm as described previously in Sagi M. and Fluhr
R. (2001 Plant Physiol 126, 1281-1290). The reaction medium
contained 6.2 .mu.g of protein extract, 1 mM epinephrine in 50 mM
Tris-HCl buffer (pH 8.5) and 1 mM hypoxanthine/xanthine. Reaction
medium without hypoxanthine/xanthine was used as control. Six
samples were analyzed and averaged for each data points.
Means.+-.SEM for each treatment are presented.
Ureides, Hypoxanthine and Xanthine Determination
[0068] Ureides were extracted from leaves with 80% ethanol and
determined according to Vogels G. D. and Van Der Drift C. (1970
Anal Biochem 33, 143-157) using allantoic acid and allantoin as
references. Six samples were analyzed and averaged for each data
points. For hypoxanthine and xanthine determination, leaves were
extracted with 40 mM NaOH and diluted four times by Tris-HCl 0.1 M,
pH 7.5. Hypoxanthine/xanthine were determined by high performance
liquid chromatography (HPLC) using a 100.times.2.1 mm ID,
ODS-Hypersil (5 .mu.m) column. The mobile phase was a 10 mM
NaOH/0.1 mM Tris-HCl, pH 5.0, similar to that used by Corpas F. J.
et al., (1997 J Plant Physiol 151, 246-250). The hypoxanthine and
xanthine were detected using standards retention time and UV
absorption spectra ratio at 249 nm and 267 nm. A modified protocol
to detect xanthine via the xanthine oxidase assay was used (Mohanty
J. G. et al., 1997 Journal of Immunological Methods 202, 133-141).
The reaction mixture modified from Yesbergenova et al., (2005
ibid), contained 0.4 U ml.sup.-1 HRP, 40 mU ml.sup.-1 buttermilk
xanthine oxidase (Fluka), 3.4 mM 3,5-dichloro-2-hydroxobenzene
sulphonate and 0.85 mM 4-aminoantipyrine in Tris-HCl 0.1 M, pH
7.5.
Example 1
XDH Expression During Dark-Induced Stress and its Subsequent
Recovery
[0069] XDH1 expression was examined under extended dark treatment
that imposes major remobilization of cellular components. To this
end, wild type Col plants and Atxdh1-compromised plants (RNA
interference lines Ri5, Ri7 and Ri10; T-DNA insertion line, KO)
impaired in XDH1 activity (Yesbergenova et al., 2005, ibid), were
exposed to 6 days continuous dark followed by a `recovery` period
of 9 days under normal diurnal light regime (16 h light/8 h dark).
The level of XDH1 transcript was found to increase continuously in
the dark reaching a peak (>20 fold induction) in 24 h (FIG. 1a,
left panel). The results imply that dark induction of XDH occurs as
part of a normal diurnal physiological response although it rises
to higher levels during the extended dark period. Upon return to
light on the 7th d the transcript level rapidly decreased (FIG. 1a,
left panel). In contrast, XDH2 transcript was unchanged during the
dark treatment although it was slightly enhanced during the
recovery period in both Col and the modified plants (FIG. 1a, right
panel).
Example 2
XDH Superoxide Activity Parallels XDH Expression
[0070] To ascertain whether potential enzymatic performance
parallels the measured transcript changes, the in-gel activities of
superoxide generation by XDH were tested using NADH and
hypoxanthine/xanthine substrates (Yesbergenova et al., 2005, ibid).
NADH-dependent activity exhibited at most a 1.5-fold enhancement in
O.sub.2.sup.- production during the dark period (FIG. 1b, left
panel), whereas the hypoxanthine/xanthine-dependent activity
increased immediately in the dark to nearly 3-fold levels at its
maximum (FIG. 1c, left panel). No NADH-dependent or
hypoxanthine/xanthine-dependent XDH activities were detected in
Atxdh1 mutants (FIGS. 1b and c, right panels; data not shown for
RNAi plants) even during the recovery period where XDH2 transcript
was observed to increase (FIG. 1a). This could indicate that XDH2
activity or abundance is very low and demonstrates that typically
XDH1 plays a role in dark stress adaptation. The results of the
in-gel assay could be confirmed in a direct crude leaf extract
assay using epinephrine as electron acceptor (Yesbergenova et al.,
2005, ibid; FIG. 1d). In this case, at least a 2-fold increase in
XDH activity could be detected in wild type leaves while mutant
lines showed no or negligible activity. In all, while the exact
fold level changes in activity differ according to the procedure
used, the measured activities followed the general rise and fall in
transcript levels.
Example 3
Mutation in XDH1 Accelerates Premature Senescence in Dark-Induced
Stressed Plants
[0071] Reference is now made to FIG. 2, in which responses of
wild-type and XDH compromised Arabidopsis plants to dark stress is
shown. In FIG. 2a, wild-type (Col) and XDH compromised plants (KO,
SALK.sub.--148364; Ri, xdh1 RNA interference) were tested during 6
day dark treatment and subsequent 9 days recovery. FIG. 2b shows
survival rates of wild-type and XDH compromised plants 15 days
after being exposed to the conditions described above. Leaves from
control untreated and dark-treated Col and Atxdh1 compromised
plants were excised before (0) and 6 d after (6) exposure to dark.
FIG. 2d shows damage level of leaves. FIG. 2e shows chlorophyll
content in leaves over a 6 d dark treatment FIG. 2f shows total
soluble protein content in leaves over a 6 d dark treatment.
[0072] Wild type Col and XDH1-compromised lines were examined more
closely during the dark treatment. Rosette leaves of the mutant
plants impaired in XDH1 expression were distinctly more yellow than
wild type leaves (FIG. 2a, c). Quantitative analysis showed a rapid
fall in residual chlorophyll levels of 30% and 50% in wild type and
mutant leaves, respectively (FIG. 2e). The yellowing was
accompanied by a reduction in soluble proteins, another hallmark of
senescence (FIG. 2f). Atxdh1-compromised leaves compared to wild
type leaves showed a higher damage rate (FIG. 2d) and accumulation
of many more dead cells as ascertained by trypan blue staining
(FIG. 2g). The plants were then returned to a normal diurnal cycle
and their subsequent recovery was followed for an additional 9 days
period. Wild-type plants showed an 80% survival rate as compared to
20 to 45% for the mutant lines (FIG. 2b). The results imply that
XDH1-dependent processes are important for plant adaptation to
extended dark treatment and subsequent recovery in normal
light.
Example 4
Senescence and Chlorophyll Degradation-Associated Gene Expression
in Atxdh1 Down-Regulated Plants
[0073] Reference is now made to FIG. 3 in which transcripts were
monitored by quantitative real-time RT-PCR analysis using wild-type
(Col), XDH compromised plants (KO, SALK.sub.--148364, Atxdh1 T-DNA
insertion line) and Ri (mean of 3 independent XDH1 RNA interference
lines).
[0074] Enhanced dark-induced leaf yellowing in the mutant lines
(such as that described in FIG. 2) may be indicative of hastened
senescence. To examine this at the molecular level, the expression
of early and late senescence-associated gene classes was monitored.
Dark stress led to more rapid and enhanced accumulation of the
early senescence transcripts, WRKY53 (senescence-related regulatory
gene) and ERD1/SAG15 (senescence-associated gene) (Brychkova et
al., 2007, ibid; Buchanan-Wollaston V. et al., 2005 Plant J 42,
567-585; Miao Y. et al., 2004, ibid) in Atxdh1 mutant leaves
compared to wild type leaves (FIG. 3, upper panels). Marker genes
that emphasize late processes in senescence are SRG1 (senescence
related gene1, a member of Fe(II)/ascorbate oxidase gene family)
and XERO1/TAS14 (dehydrin; Alsheikh M. K. et al., 2005 Plant Cell
Environ 28, 1114-1122; Brychkova et al., 2007, ibid; Canard D. et
al., 1996 Plant Physiol 112, 705-715; Weaver L. M. et al., 1998,
ibid). After 6 days they were induced 390-fold in Col wild type
plants and were yet 2-6-fold higher in the mutant lines (FIG. 3,
middle panels). Similarly, the transcript level of the Cys protease
senescence-associated gene 12 (SAG12), a common marker of leaf
senescence (Gepstein et al., 2003 Plant J 36, 629-642), exhibited
earlier and higher expression in Atxdh1 compromised plants (see
FIG. 10).
[0075] The chlorophyll-degradation gene ACD1 (accelerated cell
death1) is involved in senescence-associated chlorophyll breakdown
(Tanaka et al., 2003, ibid) while SGN1 (stay-green protein1) is
involved in chlorophyll catabolism during development (Park et al.,
2007, ibid). The transcript levels of these genes were rapidly
induced already after 1 day in darkness and were maintained at
higher levels in Atxdh1 mutants in comparison to those of Col
plants (FIG. 3, lower panels). These results indicate that the
presence of AtXDH1 plays an important role in impeding symptoms and
molecular events indicative of pre-mature senescence in response to
dark-induced stress.
Example 5
XDH and Non-XDH Superoxide Generating Activities
[0076] Reference is now made to FIG. 4 which shows quantitative
analysis of NBT-O.sub.2.sup.- production in leaves in response to
dark treatment. Plant XDH reactions can use as electron receptors
molecular oxygen yielding O.sub.2.sup.-, or if available,
non-reduced NAD.sup.+ (Hesberg C. et al., 2004 J Biol Chem 279,
13547-13554; Nguyen J. 1986 Physiologie Vegetale 24, 263-281;
Yesbergenova et al., 2005, ibid). The resultant superoxide may
further impinge on the physiology of dark-induced stress. In an
attempt to illuminate the situation in vivo, reactive oxygen
species (ROS) sources were estimated during dark treatment by the
differential use of 2 inhibitors, allopurinol and diphenylene
iodonium (DPI). The XDH inhibitor allopurinol is converted to
alloxanthine, which remains tightly attached to the
substrate-binding pocket in the MoCo domain, thereby preventing
further substrate turnover (Harrison 2002, ibid; Hesberg et al.,
2004, ibid). In contrast, DPI is a suicide inhibitor of
flavin-containing enzymes and can be used to estimate both XDH and
e.g. NADPH oxidase activities (Sagi and Fluhr, 2001, ibid;
Yesbergenova et al., 2005, ibid). To this end, rosette leaves of
dark-treated plants were examined by NBT infiltration for the
detection of superoxides (Fryer et al., 2002, ibid). During dark
treatment wild-type leaves had a basal level of total superoxide
that remained within .+-.15% of the light control (not shown). In
contrast, mutant leaves showed .about.30% lower total levels of NBT
reduction, indicating that XDH activity has a measurable
contribution to the redox milieu of the plant as has been observed
previously (Yesbergenova et al., 2005 ibid). The addition of the
XDH inhibitor, allopurinol, gives an independent measure of
XDH-dependent superoxide activity. As shown in FIG. 4b (middle
panel) a nearly 3-fold increase in XDH-dependent superoxide
production was obtained in the dark on day 3. In contrast, no such
rise was detected in XDH mutant plants. An estimate of the residual
XDH activity, due to either partial allopurinol inhibition or of
O.sub.2.sup.- production by a non-XDH source such as NADPH oxidase
activity was obtained by simultaneous subtraction of +DPI from
+allopurinol leaf scan values (FIG. 4b, lower panel). In this case
O.sub.2.sup.- production rose in the mutant plants during the dark
(i.e. the DPI inhibited activities) more than in the wild type
plants. Thus, in XDH compromised plants other sources of ROS can,
in part, compensate in the dark for the loss of ROS supplied by
XDH. Taken together, the XDH activity measured by superoxide
production increased in vivo in the dark and makes a significant
contribution to the leaf redox milieu.
Example 6
Dark Treatment Modifies Accumulation of Purine Metabolites
[0077] Reference is now made to FIG. 5 in which analysis of the
purine metabolites, xanthine, allantoin and allantoate, in response
to dark stress, is shown. It was examined whether the detected
changes in XDH1 activity impinge on the accumulation of xanthine
and/or ureides during the dark treatment and the subsequent
recovery in the light. In wild type (Col) plants the xanthine level
did not change during dark treatment over the low basal level (FIG.
5a). However, the basal level of xanthine content in Atxdh1 mutants
was more than 10-fold higher than in Col plants although
hypoxanthine levels remained below the detection limits. The
xanthine level significantly increased in Atxdh1 mutant leaves upon
dark treatment reaching over 10-fold the basal level in mutant
plants and 100-fold the level detected in wild type plants. This
level was only slightly reduced during the recovery period (FIG.
5a), indicating that potential XDH2 activity can play a typically
minor role in the control of this xanthine pool. Thus, the presence
of the XDH1 mutation unmasks a transient but significant increase
in purine catabolic flux that occurs during the dark.
[0078] XDH activity produces urate which is rapidly converted to
the ureides, allantoin and allantoate (Nguyen, 1986, ibid; Sagi et
al., 1998 ibid). Unexpectedly, their levels were found to be
significantly enhanced in wild type Col leaves upon exposing plants
to dark treatment (5-fold; FIGS. 5b, 5c, dark). The accumulation
may suggest that in wild type plants the downstream processing of
ureides is transiently blocked. Interestingly, transfer of
dark-treated wild type plants to the light after 6 days in the dark
resulted in the gradual reduction of ureides that started after the
first day and reached a basal level on the 9th day in the light
(day 15, FIGS. 5b and 5c, recovery). In Atxdh1 mutant leaves, basal
levels of allantoin and allantoate were detected and were similar
to wild type levels. Their presence may represent XDH2 activity,
residual XDH1 activity or represent an alternative pathway to
ureide biosynthesis. Importantly, the level of allantoin and
allantoate in Atxdh1 mutant leaves remained low during dark stress,
or upon recovery (FIGS. 5b, 5c). The results indicate that AtXDH1
activity plays a pivotal role in dark-induced purine catabolism by
facilitating the production of ureides.
Example 7
Dark-Induced Stress Modifies Transcript Levels of Genes Involved in
Purine Catabolism Up-Stream and Down-Stream to XDH
[0079] Reference is now made to FIG. 6 which shows quantitative
analysis of transcripts by real-time RT-PCR performed using
wild-type (Col) and XDH compromised plants (KO, SALK.sub.--148364
Atxdh1 T-DNA insertion line; Ri, means of three XDH RNA
interference lines) after being kept in the dark for 6 days and
transferred to a 16 h light/8 h dark regime for recovery during
additional 9 days. The rise in ureide levels implies a bottleneck
in their remobilization during the extended dark period, which
should be reflected in an increase or decrease of transcripts in
the catabolic pathway. AMPD, catalyzing the conversion of AMP to
IMP, is the first committed step in purine catabolism (Zrenner R.
et al., 2006 Annu Rev Plant Biol 57, 805-836). Upon transfer to
dark, AMPD transcript expression level rapidly rises in both Col
and Atxdh1 mutants and falls upon exposure to light (FIG. 6, upper
panel). In addition, transcript expression of the peroxisomal
enzymes UO and TLP, thought to be involved in ureides production
(Reumann S. et al., 2007 Plant Cell, tpc.107.050989), followed
similar pattern of transcript induction during dark stress and
subsequent recovery period, as exhibited by AMPD and XDH1 (FIG.
12). In contrast, ALN that converts allantoin to allantoate and AAH
that converts allantoate to ureidoglycolate were enhanced on day
one of the dark treatment but then declined significantly to below
the basal level on the 4th and 6th day. The latter reduction
coincides with the timing of the increase in ureides accumulation.
Upon re-exposure to light the transcripts again returned to near
basal levels (FIG. 6, middle and lower panels). Thus, transcripts
upstream and downstream from ureide biosynthesis were regulated
reciprocally. The result is consistent with the observed rise in
ureide levels and indicates that a transcriptional component exists
in the control of allantoin and allantoate accumulation during dark
treatment (FIG. 5).
Example 8
Purine Remobilization is Also Reflected in Leaf Age
[0080] Reference is made to FIG. 7, which shows the influence of
aging on leaf phenotype, purine metabolite level and transcript
expression in wild type and Atxdh1 compromised plants.
[0081] Dark treatment stimulates remobilization of metabolites
which should in part mimic processes that occur during aging. To
examine this, transcripts related to ureides synthesis were
examined in wild type and XDH1 mutant plants over the entire plant
life. With increasing age, rosette leaves of XDH mutant plants
yellowed earlier than wild-type Col plants and exhibited a
significantly higher fold level of the SAG12 transcript (FIG. 7a).
The transcripts level of enzymes leading to allantoin and
allantoate production in wild type plants, AMPD, XDH1, UO and TLP
were elevated with age. In contrast, a concomitant reduction was
observed in the transcript level of allantoin and allantoate
utilization enzymes, ALN and AAH (FIG. 7c). Consistent with these
observations, the XDH mutant showed age-dependent accumulation of
xanthine and wild type plants showed gradual accumulation of the
ureides allantoin and allantoate (FIG. 7b). Interestingly, the
levels of xanthine and ureides that accumulate with plant age were
similar to the accumulation detected during acute dark stress (FIG.
5) and may indicate the catabolic capacity of purine
metabolism.
Example 9
Role of Xanthine and Ureides in Cellular Protection During
Dark-Induced Stress
[0082] Reference is made to FIG. 8 which shows responses of
Arabidopsis wild-type (Col) and xdh RNA interference (Ri) plants to
application of the purine catabolites: xanthine, allantoin and
allantoate. The dark-induced loss of chlorophyll and the
accelerated cell death during recovery may be due to the
accumulation of excess oxypurines (FIG. 5a) or to the lack of a
protective environment provided by ureides (FIGS. 5b and 5c) or
both. To examine this directly, leaf discs cut from Col and Atxdh
mutants rosette leaves were treated with xanthine for 48 h in the
dark and were sampled after 24 h in the light. Application of 1 mM
xanthine (in the range of the calculated xanthine level that
accumulates in dark-treated mutant plants) caused a reduction in
chlorophyll which was exacerbated in the presence of allopurinol in
both wild type and mutant Ri lines (FIG. 8a). Interestingly, Col
leaf discs that exhibited the lowest chlorophyll degradation
contained enhanced allantoin and allantoate levels (FIG. 8a). The
observations indicate that ureides may protect chlorophyll from
degradation during dark treatment and the recovery period. To
elucidate the role of allantoin and allantoate directly, leaf discs
were treated with 0.1 mM allantoin or allantoate. In this case,
both types of ureides protected the treated leaf discs from
chlorophyll degradation after 48 h dark treatment (FIG. 8b).
Importantly, the protection by the ureides was afforded in spite of
the significant accumulation of xanthine in allopurinol-treated Col
plants and Atxdh mutants (FIG. 8b). These results suggest that
chlorophyll degradation resulting from dark-induced stress and
subsequent light period is due to the lack of accumulation of
ureides that likely overcome a negative role of xanthine or other
dark-induced components.
[0083] Reference is also made to FIG. 14 showing that allantoin
application prevents leaf spoilage in Parsley. Parsley leaf discs
were kept for 2 days in the light and then for 2 days in the dark
with or without the addition of 0 to 10 mM allantoin. Undetached
leaves kept in the light served as control (CL). The influence of
allantoin application on senescence in parsley was examined by
measuring the chlorophyll content relative to the content in the
control undeteched leaves. In this particular case application of
0.1-0.5 mM allantoin was particularly effective in preventing the
chlorophyll loss.
[0084] Reference is further made to FIG. 15, which shows the
response of tomato to extended dark stress and recovery in light
thereafter and the effect of application of exogenous allantoin on
leaf chlorophyll content. Allantoin level in the 1.sup.st, 3.sup.rd
and 5.sup.th leave of wild type tomato plants was measured during
12 days in the dark and during 10 days of recovery in the light
thereafter. FIG. 15a show that exposing tomato plants to dark
stress increases the allantoin level in the leaves. FIG. 15b shows
that application of allantoin to tomato leaf discs kept in the dark
for 2 days and then transferred to the light for additional two
days delayed the chlorophyll degradation.
[0085] These results show that application of 0.1-10 mM allantoin
protects leaves from chlorophyll degradation and yellowing.
According to one embodiment 0.1 mM of allantoin, 0.5 mM of
allantoin or any concentration of allantoin in this range are
sufficient to prevent yellowing.
Example 10
A Role for Ureides as Cellular Scavengers
[0086] Reference is made to FIG. 9, which shows ROS accumulation in
the presence of allantoin and allantoate in Arabidopsis wild-type
(Col) and xdh1 RNA interference (Ri) lines. ROS accumulation has
been shown to initiate chlorophyll degradation (Rentel M. C. et
al., 2004 Nature 427, 858-861) and the enhanced chlorophyll
degradation in leaves in the absence of XDH may result from ROS
production during dark stress (FIG. 4; Guo and Crawford 2005 ibid).
In light of the protective effect afforded by the ureides
application, it is possible that they act as cellular protectants
through ROS scavenging. To investigate the effect of ureides on
chlorophyll integrity and cellular scavenging capability, leaf
discs were treated with allantoin and allantoate and examined for
chlorophyll, H.sub.2O.sub.2 and superoxide levels. Remaining
chlorophyll was examined after the addition of 0.1 mM allantoin or
allantoate for 24 h and subsequent application of 20 or 50 mM
H.sub.2O.sub.2 for 6 h. The treated discs showed a significant
increase in remaining chlorophyll (26 to 31% for 20 mM
H.sub.2O.sub.2 and 32 to 36% for 50H.sub.2O.sub.2 mM, respectively;
FIG. 9a) indicating that ureides can protect leaves from ROS.
[0087] The concomitant accumulation of internal ROS as
H.sub.2O.sub.2 was examined by application of 3,3'-diaminobenzidine
(DAB). Mutant Ri lines showed 50% more DAB staining after 24 h dark
treatment (FIG. 9b). When the discs were treated with allantoin or
allantoate a significant 2-fold reduction in DAB staining intensity
was observed. NBT staining for the presence of superoxide revealed
reduced staining in Ri lines (FIG. 9c) that had been described
previously for whole leaves (FIG. 4). The addition of
allantoin/allantoate significantly reduced staining in the Col
lines but not the Ri lines. The results indicate that the presence
of ureides diminishes H.sub.2O.sub.2 accumulation and are
correlated with the observation of reduced chlorophyll degradation.
Sequence CWU 1
1
36125DNAArtificial SequenceSynthetic primer 1gattgttccc gttgcaaggt
tgttt 25228DNAArtificial SequenceSynthetic primer 2tgcaactgag
agttgtttat ggattgag 28325DNAArtificial SequenceSynthetic primer
3aagagtgggg atttttccag cttgt 25427DNAArtificial SequenceSynthetic
primer 4tgcccaatct agtttctgat cttctga 27527DNAArtificial
SequenceSynthetic primer 5tttgttaaga ctctcaccgg aaagaca
27627DNAArtificial SequenceSynthetic primer 6gagggtggat tccttctgga
tattgta 27727DNAArtificial SequenceSynthetic primer 7cactgtttat
gtgaaagcca aggaatg 27827DNAArtificial SequenceSynthetic primer
8cccaagctta aaaccatgta aatgtgg 27927DNAArtificial SequenceSynthetic
primer 9ccatgcgtta aaggaaaggt atgaaaa 271027DNAArtificial
SequenceSynthetic primer 10tgattctcag acgatcttga ggttttg
271130DNAArtificial SequenceSynthetic primer 11tcaaagaaaa
gaaagatgtt accaaagtgg 301227DNAArtificial SequenceSynthetic primer
12gtgcatctgt aataactcct tgggaat 271328DNAArtificial
SequenceSynthetic primer 13tgatgttgga caaatagaag gagcgttt
281429DNAArtificial SequenceSynthetic primer 14tattcggatt
ccccttgaga agcgaaaca 291528DNAArtificial SequenceSynthetic primer
15tgatattgga caaatagaag gagcgttt 281629DNAArtificial
SequenceSynthetic primer 16tgcatttgga ttacccttga gaagagaaa
291726DNAArtificial SequenceSynthetic primer 17agactcacca
acagcttgac caattt 261826DNAArtificial SequenceSynthetic primer
18cacctagtcc atcatccgag ctagag 261925DNAArtificial
SequenceSynthetic primer 19gattgttccc gttgcaaggt tgttt
252028DNAArtificial SequenceSynthetic primer 20tgcaactgag
agttgtttat ggattgag 282125DNAArtificial SequenceSynthetic primer
21aagagtgggg atttttccag cttgt 252227DNAArtificial SequenceSynthetic
primer 22tgcccaatct agtttctgat cttctga 272327DNAArtificial
SequenceSynthetic primer 23tttgttaaga ctctcaccgg aaagaca
272427DNAArtificial SequenceSynthetic primer 24gagggtggat
tccttctgga tattgta 272527DNAArtificial SequenceSynthetic primer
25cactgtttat gtgaaagcca aggaatg 272627DNAArtificial
SequenceSynthetic primer 26cccaagctta aaaccatgta aatgtgg
272727DNAArtificial SequenceSynthetic primer 27ccatgcgtta
aaggaaaggt atgaaaa 272827DNAArtificial SequenceSynthetic primer
28tgattctcag acgatcttga ggttttg 272930DNAArtificial
SequenceSynthetic primer 29tcaaagaaaa gaaagatgtt accaaagtgg
303027DNAArtificial SequenceSynthetic primer 30gtgcatctgt
aataactcct tgggaat 273128DNAArtificial SequenceSynthetic primer
31tgatgttgga caaatagaag gagcgttt 283229DNAArtificial
SequenceSynthetic primer 32tattcggatt ccccttgaga agcgaaaca
293328DNAArtificial SequenceSynthetic primer 33tgatattgga
caaatagaag gagcgttt 283429DNAArtificial SequenceSynthetic primer
34tgcatttgga ttacccttga gaagagaaa 293526DNAArtificial
SequenceSynthetic primer 35agactcacca acagcttgac caattt
263626DNAArtificial SequenceSynthetic primer 36cacctagtcc
atcatccgag ctagag 26
* * * * *