U.S. patent application number 09/834998 was filed with the patent office on 2002-11-28 for proton transporters and uses in plants.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. Invention is credited to Alper, Seth L., Fink, Gerald R., Gaxiola, Roberto A..
Application Number | 20020178464 09/834998 |
Document ID | / |
Family ID | 26860868 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020178464 |
Kind Code |
A1 |
Gaxiola, Roberto A. ; et
al. |
November 28, 2002 |
Proton transporters and uses in plants
Abstract
The present invention relates to a transgenic plant which is
tolerant to a salt, comprising one or more plant cells transformed
with exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant. Also encompassed by the present
invention are transgenic progeny and seeds of the transgenic plants
described herein. Progeny transgenic plant grown from seed are also
described. The present invention also relates to a construct
comprising an AVP1 gene operably linked to a chimeric promoter
designed to overexpress AVP1 or designed to down regulate
endogenous pyrophosphatase. Plant cells (e.g., root cells, stem
cell, leaf cells) comprising exogenous nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant cell are also
the subject of the present invention. Also encompassed by the
present invention are methods of making a transgenic plant
described herein. Transgenic plants produced by the methods of
making a transgenic plant as described herein are also a subject of
the present invention. The present invention also relates to a
method of bioremediating soil, a method of increasing the yield of
a plant, a method of making a plant which is larger than its
corresponding wild type plant, and a method of producing a
transgenic plant which grows in salt water comprising introducing
into one or more cells of a plant nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant. The transgenic
plants of the present invention can also be used to produce double
transgenic plants which are tolerant to a salt.
Inventors: |
Gaxiola, Roberto A.;
(Mansfield Center, CT) ; Fink, Gerald R.;
(Chestnut Hill, MA) ; Alper, Seth L.; (Boston,
MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Whitehead Institute for Biomedical
Research
Nine Cambridge Center
Cambridge
MA
02142
|
Family ID: |
26860868 |
Appl. No.: |
09/834998 |
Filed: |
April 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09834998 |
Apr 13, 2001 |
|
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09644039 |
Aug 22, 2000 |
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60164808 |
Nov 10, 1999 |
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Current U.S.
Class: |
800/278 ;
800/317.3; 800/317.4; 800/320.1 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8261 20130101; Y02A 40/146 20180101; C12N 15/8242 20130101;
C12N 15/8273 20130101 |
Class at
Publication: |
800/278 ;
800/317.3; 800/320.1; 800/317.4 |
International
Class: |
A01H 005/00 |
Goverment Interests
[0002] The invention was supported, in whole or in part, by grants
GM52414, DK54214, DK43495, DK51509, DK34854 and GM35010 from the
National Institutes of Health and by grant MCB9317175 from the
National Science Foundation. The Government has certain rights in
the invention.
Claims
What is claimed is:
1. A transgenic plant which is tolerant to a salt, comprising one
or more plant cells transformed with exogenous nucleic acid which
alters expression of vacuolar pyrophosphatase in the plant.
2. The transgenic plant of claim 1 wherein the exogenous nucleic
acid encodes AVP1 or a homologue thereof.
3. The transgenic plant of claim 2 wherein the homologue of AVP1 is
obtained from tobacco, bacteria, tomato or corn.
4. The transgenic plant of claim 2 wherein the AVP1 is present in a
construct designed to overexpress AVP1 or designed to down regulate
endogenous pyrophosphatase.
5. The transgenic plant of claim 4 wherein the construct comprises
AVP1 operably linked to a double tandem enhancer of a 35S
promoter.
6. The transgenic plant of claim 2 wherein the AVP1 is derived from
a wild type plant that corresponds to a wild type of the transgenic
plant.
7. The transgenic plant of claim 2 wherein the AVP1 is derived from
a wild type plant that does not correspond to a wild type of the
transgenic plant .
8. A transgenic plant which grows in a concentration of a salt that
inhibits growth of a corresponding non-transgenic plant.
9. The transgenic plant of claim 8 wherein the concentration of
salt is about 0.2M to about 0.3M.
10. The transgenic plant of claim 1 wherein the plant is larger
than a corresponding non-transgenic plant.
11. A transgenic progeny of the transgenic plant of claim 1.
12. Seeds produced by the transgenic plant of claim 1.
13. A progeny transgenic plant grown from seed of claim 12.
14. A transgenic plant which is tolerant to a salt comprising an
exogenous nucleic acid construct which is designed to overexpresses
AVP1 or designed to down regulate endogenous pyrophosphatase.
15. A transgenic progeny of the transgenic plant of claim 14.
16. Seeds produced by the transgenic plant of claim 14.
17. A progeny transgenic plant grown from seed of claim 16.
18. The transgenic plant of claim 14 wherein the construct
comprises an AVP1 gene operably linked to a double tandem enhancer
of a 35S promoter.
19. A construct comprising an AVP1 gene operably linked to a
chimeric promoter designed to overexpress AVP1 or designed to down
regulate endogenous pyrophosphatase.
20. The construct of claim 19 wherein the AVP1 gene is operably
linked to a double tandem enhancer of a 35S promoter.
21. A transgenic plant obtained by introducing into a plant
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant.
22. Plant cells comprising exogenous nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant cell.
23. The plant cells of claim 22 wherein the plant cells are root
cells or stem cells.
24. The plant cells of claim 22 wherein the exogenous nucleic acid
encodes AVP1.
25. The plant cells of claim 24 wherein the AVP1 is present in a
construct designed to overexpress AVP1 or designed to down regulate
endogenous pyrophosphatase.
26. The plant cells of claim 25 wherein the construct comprises the
AVP1 gene operably linked to a chimeric promoter designed to
overexpress AVP1.
27. The plant cells of claim 26 wherein the AVP1 gene is operably
linked to a double tandem enhancer of a 35S promoter.
28. The plant cells of claim 24 wherein the AVP1 is derived from a
wild type plant that corresponds to a wild type of the transgenic
plant.
29. The plant cells of claim 24 wherein the AVP1 is derived from a
wild type plant that does not correspond to a wild type of the
transgenic plant.
30. A method of making a transgenic plant which is tolerant to a
salt comprising introducing into one or more cells of a plant
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant to yield transformed cells in the
plant, thereby producing a transgenic plant which is tolerant to
the salt.
31. The method of claim 30 further comprising regenerating plants
from the transformed cells to yield transgenic plants and selecting
a transgenic plant which is tolerant to the salt, thereby producing
a transgenic plant which is tolerant to the salt.
32. The method of claim 30 wherein the exogenous nucleic acid
encodes AVP1.
33. The method of claim 32 wherein the AVP1 is present in a
construct designed to overexpress AVP1 or designed to down regulate
endogenous pyrophosphatase.
34. The method of claim 33 wherein the construct comprises the AVP1
gene operably linked to a chimeric promoter designed to overexpress
AVP1.
35. The method of claim 34 wherein the AVP1 gene is operably linked
to a double tandem enhancer of a 35S promoter.
36. The method of claim 32 wherein the AVP1 is derived from a wild
type plant that corresponds to a wild type of the transgenic
plant.
37. The method of claim 32 wherein the AVP1 is derived from a wild
type plant that does not correspond to a wild type of the
transgenic plant.
38. The method of claim 30 wherein the plant is tolerant to a
concentration of salt that inhibits growth of a corresponding
non-transgenic plant.
39. The transgenic plant of claim 38 wherein the concentration of
salt is about 0.2M to about 0.3M.
40. A transgenic plant produced by the method of claim 30.
41. A method of making a transgenic plant which is tolerant to a
salt comprising introducing into one or more cells of a plant a
nucleic acid construct which is designed to overexpress AVP1 to
yield transformed cells thereby producing a transgenic plant which
is tolerant to the salt.
42. The method of claim 41 further comprising regenerating plants
from the transformed cells to yield transgenic plants and selecting
a transgenic plant which is tolerant to the salt, thereby producing
a transgenic plant which is tolerant to the salt.
43. A transgenic plant produced by the method of claim 41.
44. A method of making a transgenic plant which is larger than its
corresponding wild type plant comprising introducing into one or
more cells of a plant nucleic acid which alters expression of
vacuolar pyrophosphatase in the plant to yield transformed cells,
thereby producing a transgenic plant which is larger than its
corresponding wild type plant.
45. The method of claim 44 further comprising regenerating plants
from the transformed cells to yield transgenic plants and selecting
a transgenic plant which is larger than its corresponding wild type
plant, thereby producing a transgenic plant which is larger than
its corresponding wild type plant.
46. The method of claim 44 wherein the exogenous nucleic acid
encodes AVP1.
47. The method of claim 46 wherein the AVP1 is present in a
construct designed to overexpress AVP1 or designed to down regulate
endogenous pyrophosphatase.
48. The method of claim 47 wherein the construct comprises the AVP1
gene operably linked to a chimeric promoter designed to overexpress
AVP1.
49. The method of claim 48 wherein the AVP1 gene is operably linked
to a double tandem enhancer of a 35S promoter.
50. The method of claim 46 wherein the AVP1 is derived from a wild
type plant that corresponds to a wild type of the transgenic
plant.
51. The method of claim 46 wherein the AVP1 is derived from a wild
type plant that does not correspond to a wild type of the
transgenic plant.
52. The method of claim 44 wherein the transgenic plant is grown in
soil.
53. The method of claim 44 wherein the transgenic plant is grown
hydroponically.
54. A transgenic plant produced by the method of claim 44.
55. A method of bioremediating soil comprising growing one or more
transgenic plants and/or progeny thereof in the soil, wherein the
transgenic plants and/or progeny thereof comprise exogenous nucleic
acid which alters expression of vacuolar pyrophosphatase in the
plant.
56. A method of increasing the yield of a plant comprising
introducing into one or more cells of a plant nucleic acid which
alters expression of vacuolar pyrophosphatase in the plant to yield
transformed cells, thereby increasing the yield of the plant.
57. The method of claim 56 further comprising regenerating plants
from the transformed cells to yield transgenic plants and selecting
a transgenic plant which is larger than its corresponding wild type
plant, thereby increasing the yield of the plant.
58. A method of removing cations from a medium which can support
plant growth comprising growing one or more transgenic plants
and/or progeny thereof in the medium, wherein the transgenic plants
and/or progeny thereof comprise exogenous nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant.
59. The method of claim 58 wherein the medium is selected from the
group consisting of: soil and water.
60. The method of claim 58 wherein the cations are selected from
the group consisting of: sodium, calcium, manganese and lead.
61. A method of producing a transgenic plant which grows in salt
water comprising introducing into one or more cells of a plant
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant to yield transformed cells, thereby
producing a transgenic plant which can grow in salt water.
62. The method of claim 61 further comprising regenerating plants
from the transformed cells to yield transgenic plants and selecting
a transgenic plant which grow in salt water, thereby producing a
transgenic plant which can grow in salt water.
63. The method of claim 61 wherein the concentration of the salt
water is from about 0.2M to about 0.3M.
64. The method of claim 63 wherein the salt water is seawater.
65. A transgenic plant which is tolerant to a salt, comprising one
or more plant cells transformed with exogenous nucleic acid which
alters expression of a vacuolar pyrophosphatase and an
Na.sup.+/H.sup.+ antiporter in the plant.
66. The transgenic plant of claim 65 wherein the vacuolar
pyrophosphatase is AVP1 or a homologue thereof.
67. The transgenic plant of claim 66 wherein the homologue of AVP1
is from tobacco, bacteria, tomato or corn.
68. The transgenic plant of claim 66 wherein the AVP1 is present in
a construct designed to overexpress AVP1 or designed to down
regulate endogenous pyrophosphatase.
69. The transgenic plant of claim 68 wherein the construct
comprises AVP1 operably linked to a double tandem enhancer of a 35S
promoter.
70. The transgenic plant of claim 65 wherein the Na.sup.+/H.sup.+
antiporter is AtNHX1 or a homologue thereof.
71. A transgenic progeny of the transgenic plant of claim 65.
72. Seeds produced by the transgenic plant of claim 65.
73. A progeny transgenic plant grown from the seed of claim 72.
74. A method of making a transgenic plant having increased flower
size compared to its corresponding wild type plant comprising
introducing into one or more cells of a plant nucleic acid which
alters expression of vacuolar pyrophosphatase in the plant to yield
transformed cells, thereby producing a transgenic plant having
increased flower size compared to its corresponding wild type
plant.
75. The method of claim 74 wherein the exogenous nucleic acid
encodes AVP1.
76. A transgenic plant produced by the method of claim 74.
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 09/644,039, entitled "Proton Transporters and Uses in Plants"
by Roberto A. Gaxiola, filed Aug. 22, 2000, which claims the
benefit of U.S. application Ser. No. 60/164,808, entitled "Proton
Transporters and Uses in Plants" by Roberto Gaxiola, filed Nov. 10,
1999. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The prospects for feeding humanity as we enter the new
millennium are formidable. The progressive salinization of
irrigated land compromises the future of agriculture in the most
productive areas of our planet (Serrano, R., et al., Crit. Rev.
Plant Sci., 13:121-138 (1994)). Arid regions offer optimal
photoperiod and temperature conditions for the growth of most
crops, but suboptimal rainfall. Artificial irrigation has solved
the problem in the short term. However, water supplies always
contain some dissolved salt, which upon evaporation gradually
accumulates on the soils.
[0004] To grow in saline environments, plants must maintain a much
lower ratio of Na.sup.+/K.sup.+ in their cytoplasm than that
present in the soil. A need exists for crops having increased
tolerance to salt.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a transgenic plant which is
tolerant to a salt, comprising one or more plant cells transformed
with exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant. In one embodiment, the exogenous
nucleic acid encodes AVP1 or a homologue thereof. In another
embodiment, the present invention relates to a transgenic plant
which grows in a concentration of a salt that inhibits growth of a
corresponding non-transgenic plant. In a particular embodiment, the
transgenic plant is tolerant to sodium chloride (NaCl) and the NaCl
concentration is from about 0.2M to about 0.3M. In a another
embodiment, the present invention relates to a transgenic plant
which is tolerant to salt comprising (containing within some or all
of its cells) an exogenous nucleic acid construct which is designed
to overexpress AVP1 or designed to down regulate endogenous
pyrophosphatase. In yet another embodiment, the invention relates
to a transgenic plant obtained by introducing into a plant
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant.
[0006] Also encompassed by the present invention are transgenic
progeny and seeds of the transgenic plants described herein.
Progeny transgenic plants grown from seed of transgenic plants are
also described.
[0007] The present invention also relates to a construct comprising
an AVP1 gene operably linked to a chimeric promoter designed to
overexpress AVP1 or designed to down regulate endogenous
pyrophosphatase. In one embodiment, the AVP1 gene is operably
linked to a double tandem enhancer of a 35S promoter.
[0008] Plant cells (e.g., root cells, stem cell, leaf cells)
comprising exogenous nucleic acid which alters expression of
vacuolar pyrophosphatase in the plant cell are also the subject of
the present invention.
[0009] Also encompassed by the present invention is a method of
making a transgenic plant which is tolerant to salt comprising
introducing into one or more cells of a plant exogenous nucleic
acid which alters expression of vacuolar pyrophosphatase in the
plant to yield transformed cells in the plant, thereby producing a
transgenic plant which is tolerant to salt. For example, this can
be carried out in a whole plant, seeds, leaves, roots or any other
plant part. In one embodiment, the present invention relates to a
method of making a transgenic plant which is tolerant to salt
comprising introducing into one or more cells of a plant a nucleic
acid construct which is designed to overexpress AVP1 to yield
transformed cells thereby producing a transgenic plant which is
tolerant to salt. The method can further comprise regenerating
plants from the transformed cells to yield transgenic plants and
selecting a transgenic plant which is tolerant to salt, thereby
producing a transgenic plant which is tolerant to salt.
[0010] The present invention also relates to a method of making a
transgenic plant which is larger than its corresponding wild type
plant comprising introducing into one or more cells of a plant
nucleic acid which alters expression of vacuolar pyrophosphatase in
the plant to yield transformed cells, thereby producing a
transgenic plant which is larger than its corresponding wild type.
The method can further comprise regenerating plants from the
transformed cells to yield transgenic plants and selecting a
transgenic plant which is tolerant to salt, thereby producing a
transgenic plant which is larger than its corresponding wild
type.
[0011] Transgenic plants produced by the methods of making a
transgenic plant as described herein are also a subject of the
present invention.
[0012] The present invention relates to a method of bioremediating
soil comprising growing one or more transgenic plants and/or
progeny thereof in the soil, wherein the transgenic plants and/or
progeny thereof comprise exogenous nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant. The transgenic
plants grow in the soil, and in the process, take up cations from
the soil. In one embodiment, the present invention relates to a
method of removing one or more cations from a medium which can
support plant growth (e.g., soil, water) comprising growing one or
more transgenic plants and/or progeny thereof in the medium,
wherein the transgenic plants and/or progeny thereof comprise
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant.
[0013] The present invention provides for a method of increasing
the yield of a plant comprising introducing into one or more cells
of a plant nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant to yield transformed cells, thereby
increasing the yield of the plant. The method can further comprise
regenerating plants from the transformed cells to yield transgenic
plants and selecting a transgenic plant which is larger than its
corresponding wild type plant, thereby producing a transgenic plant
which is larger than its corresponding wild type plant.
[0014] Also encompassed by the present invention is a method of
making a transgenic plant (e.g., an ornamental plant) having
increased flower size compared to its corresponding wild type plant
comprising introducing into one or more cells of a plant nucleic
acid which alters expression of vacuolar pyrophosphatase in the
plant to yield transformed cells, thereby producing a transgenic
plant having increased flower size compared to its corresponding
wild type plant.
[0015] The present invention also provides for a method of
producing a transgenic plant which grows in salt water, such as
water in which the salt concentration is equivalent to that of
seawater (e.g., about 0.2M to about 0.4M), comprising introducing
into one or more cells of a plant nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant to yield
transformed cells, thereby producing a transgenic plant which grows
in salt water. The method can further comprise regenerating plants
from the transformed cells to yield transgenic plants and selecting
a transgenic plant which is larger than its corresponding wild type
plant, thereby producing a transgenic plant which can grow in salt
water.
[0016] The transgenic plants of the present invention can also be
used to produce double transgenic plants which are tolerant to salt
(about 0.2M to about 0.4M salt concentration). In one embodiment,
the present invention relates to a double transgenic plant which is
tolerant to salt comprising one or more plant cells transformed
with exogenous nucleic acid which alters expression of a vacuolar
pyrophosphatase and an Na.sup.+/H.sup.+antiporter in the plant. In
one embodiment, the vacuolar pyrophosphatase is AVP1 or a homologue
thereof and the Na.sup.+/H.sup.+antiporter is AtNHX1 or a homologue
thereof. The present invention further relates to a transgenic
progeny of the double transgenic plant, as well as seeds produced
by the transgenic plant and a progeny transgenic plant grown from
the seed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A and 1B are bar graphs showing the intracellular
Na.sup.+ and K.sup.+ contents of wild-type yeast strains and of
yeast strains carrying various mutations affecting sodium
tolerance; values are the mean of two determinations, and bars
represent the standard deviations.
[0018] FIG. 2 is alignment of the deduced amino acid sequences of
NhXl homologue from Arabidopsis AtNHX1 (SEQ ID NO: 1), human
HsNHE-6 (SEQ ID NO:2) and yeast ScNHX1 (SEQ ID NO:3); identical
residues are in black boxes, and dashes indicate gaps in the
sequence, * above alignment denote putative amiloride binding site
from human NHE1 (.sup.163DVF-FLFLLPPI.sup- .173) (SEQ ID NO:
4).
[0019] FIG. 3A is a schematic representation of a working model of
the transporters involved in sodium sequestration at the yeast
prevacuolar compartment; Nhx1 (Na.sup.+/H.sup.+ antiporter), Vmal
(vacuolar membrane H.sup.+-ATPAse), Gef1 (yeast CLC chloride
channel), Enal (plasma membrane Na.sup.+-ATPase).
[0020] FIG. 3B is a schematic representation of a working model of
the transporters involved in sodium sequestration at the yeast
prevacuolar compartment shown in FIG. 3A, which also includes Avp1
(A. thaliana vacuolar pyrophosphate-energized proton pump).
DETAILED DESCRIPTION OF THE INVENTION
[0021] Producing salt-tolerant plants using genetic engineering
requires the identification of the relevant genes. Physiological
studies suggest that salt exclusion in the root and/or salt
sequestration in the leaf cell vacuoles are critical determinants
for salt tolerance (Kirsch, M., et al., Plant Mol. Biol.,
32:543-547 (1996)). Toxic concentrations of sodium chloride (NaCl)
build up first in the fully expanded leaves where NaCl is
compartmentalized in the vacuoles. Only after their loading
capacity is surpassed, do the cytosolic and apoplasmic
concentrations reach toxic levels, ultimately leading to loss of
turgor, ergo plant death. It has been suggested that
hyperacidification of the vacuolar lumen via the V-ATPase provides
the extra protons required for a Na.sup.+/H.sup.+ exchange-activity
leading to the detoxification of the cytosol (Tsiantis, M. S., et
al, Plant J., 9:729-736 (1996)). Salt stress increases both ATP-
and pyrophosphate (PPi)-dependent H.sup.+ transport in tonoplast
vesicles from sunflower seedling roots. Salt treatments also induce
an amiloride-sensitive Na.sup.+/H.sup.+ exchange activity
(Ballesteros, E., et al., Physiologia Plantarum, 99:328-334
(1997)). In the halophyte Mesembryanthemum crystallinum, high NaCl
stimulates the activities of both the vacuolar H.sup.+-ATPase
(V-ATPase) and a vacuolar Na.sup.+/H.sup.+ antiporter in leaf
cells. As described herein, the plant components involved in the
intracellular detoxification system have been identified by
complementing salt-sensitive mutants of the budding yeast
Saccharomyces cerevisiae. As also described herein, Arabidopsis
thaliana has been used as a host model plant to demonstrate that
overexpression of these genes results in salt tolerance in the
plant.
[0022] Accordingly, the present invention is directed to transgenic
plants which are tolerant to one or more salts. As used herein, the
term "salt" refers to any salt, such as NaCl, KCl, and/or
CaCl.sub.2. In one embodiment, the transgenic plants of the present
invention comprise one or more plant cells transformed with
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant. Any suitable vacuolar
pyrophosphatase, several of which have been cloned, can be used in
the compositions and methods of the present invention (e.g.,
Sarasian, Z., et al., Proc. Natl. Acad. Sci., USA, 89:1775-1779
(1992); Jenslerchl, et al., Molec. Biol., 29: 833-840 (1995); Kim,
Y., et al., Plant Physiol., 106:375-382 (1994)). As used herein,
nucleic acid which "alters expression of vacuolar pyrophosphatase"
includes nucleic acid which enhances (promotes) or inhibits
expression of vacuolar pyrophosphatase in the transgenic plant. In
a particular embodiment, the present invention relates to a
transgenic plant which is tolerant to salt comprising an exogenous
nucleic acid construct which is designed to overexpress AVP1
(Sarasian, Z., et al., Proc. Natl. Acad. Sci., USA, 89:1775-1779
(1992)) or designed to downregulate endogenous vacuolar
pyrophosphatase. The present invention also encompasses transgenic
plants which grow in a concentration of salt that inhibits growth
of a corresponding non-transgenic plant. Transgenic progeny of the
transgenic plants, seeds produced by the transgenic plant and
progeny transgenic plants grown from the transgenic seed are also
the subject of the present invention. Also described herein are
plant cells comprising exogenous nucleic acid which alters
expression of vacuolar pyrophosphatase in the plant cell.
[0023] Any suitable nucleic acid molecule which alters expression
of vacuolar pyrophosphatase in the plant can be used to transform
the transgenic plants in accordance with the present invention.
Exogenous nucleic acid is a nucleic acid from a source other than
the plant cell into which it is introduced or into a plant or plant
part from which the tansgenic part was produced. The exogenous
nucleic acid used for transformation can be RNA or DNA, (e.g.,
cDNA, genomic DNA). In addition, the exogenous nucleic acid can be
circular or linear, double-stranded or single-stranded molecules.
Single-stranded nucleic acid can be the sense strand or the
anti-sense strand.
[0024] The exogenous nucleic acid can comprise nucleic acid that
encodes a vacuolar pyrophosphatase protein (an exogenous vacuolar
pyrophosphatase), such as AVP1, a functional portion thereof
(peptide, polypeptide), or a homologue thereof, and/or nucleic acid
that alters (enhances, inhibits) expression of the endogenous
vacuolar pyrophosphatase of the plant into which the exogenous
nucleic acid is introduced. As used herein a "functional portion"
of a nucleic acid that encodes a vacuolar pyrophosphatase protein
is a portion of the nucleic acid that encodes a protein or
polypeptide which retains a function characteristic of a vacuolar
pyrophosphatase protein. In a particular embodiment, the nucleic
acid encodes AVP1, a functional portion or a homologue thereof.
[0025] Nucleic acid that alters (enhances, inhibits) expression of
the endogenous vacuolar pyrophosphatase of the plant into which the
exogenous nucleic acid is introduced includes regulatory sequences
(e.g., inducible, constitutive) which function in plants and
antisense nucleic acid. Examples of regulatory sequences include
promoters, enhancers and/or suppressors of vacuolar
pyrophosphatase. The nucleic acid can also include, for example,
polyadenylation site, reporter gene and/or intron sequences and the
like whose presence may not be necessary for function or expression
of the nucleic acid but can provide improved expression and/or
function of the nucleic acid by affecting, for example,
transcription and/or stability (e.g., of mRNA). Such elements can
be included in the nucleic acid molecule to obtain optimal
performance of the nucleic acid.
[0026] The nucleic acid for use in the present invention can be
obtained from a variety sources using known methods. For example,
the nucleic acid encoding a vacuolar pyrophosphatase (e.g., AVP1)
for use in the present invention can be derived from a natural
source, such as tobacco, bacteria, tomato or corn. In one
embodiment, the nucleic acid encodes a vacuolar pyrophosphatase
that corresponds to a wild type of the transgenic plant. In another
embodiment, the nucleic acid encodes a vacuolar pyrophosphatase
that does not correspond to a wild type of the transgenic plant.
Nucleic acid that alters (enhances, inhibits) expression of the
endogenous vacuolar pyrophosphatase of the plant into which the
exogenous nucleic acid is introduced (e.g., regulatory sequence)
can also be chemically synthesized, recombinantly produced and/or
obtained from commercial sources.
[0027] A variety of methods for introducing the nucleic acid of the
present invention into plants are known to those of skill in the
art. For example, Agrobacteriummediated plant transformation,
particle bombardment, microparticle bombardment (e.g., U.S. Pat.
No. 4,945,050; U.S. Pat. No. 5,100,792) protoplast transformation,
gene transfer into pollen, injection into reproductive organs and
injection into immature embryos can be used. The exogenous nucleic
acid can be introduced into any suitable cell(s) of the plant, such
a root cell(s), stem cell(s) and/or leaf cell(s) of the plant.
[0028] In one embodiment, a construct comprising a vacuolar
pyrophosphatase gene operably linked to a promoter designed to
overexpress the vacuolar pyrophosphatase (e.g., an expression
cassette) or a construct designed to downregulate endogenous
pyrophosphatase is used to produce the transgenic plants of the
present invention. As used herein the term "overexpression" refers
to greater expression/activity than occurs in the absence of the
construct. In a particular embodiment, a construct comprising an
AVP1 gene operably linked to a chimeric promoter designed to
overexpress the AVP1 or designed to downregulate endogenous
pyrophosphatase is used to produce the transgenic plants of the
present invention. More particularly, the present invention relates
to a construct wherein the AVP1 gene is operably linked to a double
tandem enhancer of a 35S promoter.
[0029] Any suitable plant can be used to produce the transgenic
plants of the present invention. For example, tomato, corn,
tobacco, rice, sorghum, cucumber, lettuce, turf grass, ornamental
(e.g., larger flowers, larger leaves) and legume plants can be
transformed as described herein to produce the transgenic plants of
the present invention. In addition, the transgenic plants of the
present invention can be grown in any medium which supports plant
growth such as soil or water (hydroponically).
[0030] The present invention also encompasses methods of making a
transgenic plant which is tolerant to salt. In one embodiment, the
method comprises introducing into one or more cells of a plant
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant to yield transformed cells in the
plant, thereby producing a transgenic plant which is tolerant to
salt. In another embodiment, the method comprises introducing into
one or more cells of a plant a nucleic acid construct which is
designed to overexpress AVP1 to yield transformed cells, thereby
producing a transgenic plant which is tolerant to salt. The methods
of making a transgenic plant can further comprise regenerating
plants from the transformed cells to yield transgenic plants and
selecting a transgenic plant which is tolerant to salt. The
transgenic plants produced by these methods are also encompassed by
the present invention.
[0031] The transgenic plants of the present invention are useful
for a variety of purposes. As described herein, the plant
components involved in an intracellular cation detoxification
system have been identified by complementing salt-sensitive mutants
of the budding yeast Saccharomyces cerevisiae. The present
invention relates to a method of bioremediating soil comprising
growing one or more transgenic plants and/or progeny thereof in the
soil, wherein the transgenic plants and/or progeny thereof comprise
exogenous nucleic acid which alters expression of vacuolar
pyrophosphatase in the plant. In another embodiment, the present
invention relates to a method of removing cations (e.g., monvalent
and/or divalent cations) from a medium which can support plant
growth (e.g., soil, water) comprising growing one or more
transgenic plants and/or progeny thereof in the medium, wherein the
transgenic plants and/or progeny thereof comprise exogenous nucleic
acid which alters expression of vacuolar pyrophosphatase in the
plant. For example, the method can be used to remove sodium (Na),
lead (Pb), manganese (Mn) and/or calcium (Ca) ions from a medium
which supports plant growth.
[0032] Furthermore, it has been shown herein that the transgenic
plants of the present invention are larger than the corresponding
wild type plants (Example 3). Thus, the present invention provides
for a method of increasing the yield of a plant comprising
introducing into one or more cells of a plant nucleic acid which
alters expression of vacuolar pyrophosphatase in the plant to yield
transformed cells, thereby increasing the yield of the plant. The
present invention also relates to a method of making a plant which
is larger than its corresponding wild type plant comprising
introducing into one or more cells of a plant nucleic acid which
alters expression of vacuolar pyrophosphatase in the plant to yield
transformed cells, thereby producing a transgenic plant which is
larger than its corresponding wild type plant. The method can
further comprise regenerating plants from the transformed cells to
yield transgenic plants and selecting a transgenic plant which is
larger than its corresponding wild type plant, thereby producing a
transgenic plant which is larger than its corresponding wild type
plant. Also encompassed by the present invention is a method of
making a transgenic plant (e.g., an ornamental plant) having
increased flower size compared to its corresponding wild type plant
comprising introducing into one or more cells of a plant nucleic
acid which alters expression of vacuolar pyrophosphatase in the
plant to yield transformed cells, thereby producing a transgenic
plant having increased flower size compared to its corresponding
wild type plant.
[0033] The present invention also provides for a method of
producing a transgenic plant which grows in salt water comprising
introducing into one or more cells of a plant nucleic acid which
alters expression of vacuolar pyrophosphatase in the plant to yield
transformed cells, thereby producing a transgenic plant which grows
in salt water. As used herein, "salt water" includes water
characterized by the presence of salt, and preferably wherein the
concentration of salt in the water is from about 0.2M to about
0.4M. In one embodiment, salt water refers to sea water.
[0034] The transgenic plants of the present invention can also be
used to produce double transgenic plants which are tolerant to salt
wherein a plant is transformed with exogenous nucleic acid which
alters expression of a vacuolar phosphatase and exogenous nucleic
acid which alters expression of another protein involved in
sequestration of cations and/or detoxification in plants. In one
embodiment, the present invention relates to a double transgenic
plant which is tolerant to salt comprising one or more plant cells
transformed with exogenous nucleic acid which alters expression of
a vacuolar pyrophosphatase and an Na.sup.+/H.sup.+ antiporter in
the plant. In one embodiment, the vacuolar pyrophosphatase is AVP1
or a homologue thereof and the Na.sup.+/H.sup.+ antiporter is
AtNHX1 or a homologue thereof. The present invention further
relates to a transgenic progeny of the double transgenic plant, as
well as seeds produced by the transgenic plant and a progeny
transgenic plant grown from the seed.
[0035] Investigation of the role of intracellular organelles in
cation homeostasis via the identification and manipulation of key
transporters is described herein. Most of these intracellular
organelles, including clathrin-coated vesicles, endosomes, Golgi
membranes and vacuoles have acidic interiors (Xie, X. S., et al.,
J. Biol. Chem., 264:18870-18873 (1989)). This acidification is
mediated by a proton-translocating electrogenic ATPase and in plant
vacuoles also via a pyrophosphate-driven proton pump V-PPase
(Davies, J. M., et al., The Bioenergetics of Vacuolar H.sup.+
Pumps. In: Leigh R. A., Sanders, D., (eds) The Plant Vacuole, pp.
340-363, Academic Press, San Diego (1997); Zhen, R. G., et al.,
"The Molecular and Biochemical Basis of Pyrophosphate-Energized
Proton Translocation at the Vacuolar Membrane Academic Press
Limited (1997)). There exists a requirement of anion transport to
maintain net electroneutrality (al-Awqati, A., Curr. Opin. Cell.
Biol., 7:504-508 (1995)). The yeast member of the CLC voltage-gated
chloride channel superfamily, Gef1, is required for copper loading
in late-Golgi vesicles and for cation sequestration in the
prevacuolar compartment in yeast (Gaxiola, R. A., et al., Proc.
Natl. Acad. Sci. USA, 95:4046-4050 (1998); Gaxiola, R. A., et al.,
Proc. Natl. Acad. Sci. USA, 96:1480-1485 (1999); Example 1).
Furthermore, it has been shown that the defects of gef1 mutants can
be suppressed by the introduction of the prototype member of the
CLC superfamily, the Torpedo marmorata CLC-0 or by the introduction
of Arabidobsis thaliana CLC-c and CLC-d chloride channel genes
(Hechenberger, M., et al., J. Biol. Chem., 271:33632-33638 (1996);
Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050
(1998)). While not wishing to be bound by theory, two observations
led to the proposal of a model for Na.sup.+ sequestration in yeast
described herein (FIGS. 3A and 3B). First, gef1 mutants are
sensitive to high NaCl concentrations. Second, the
Na.sup.+/H.sup.+exchanger Nhx1 localized to the prevacuolar
compartment (Nass, R., et al., J Biol. Chem., 273:21054-21060
(1998)). This model posits that Na.sup.+ sequestration by Nhx1
depends on the vacuolar H.sup.+-ATPase and Gef1, the chloride
channel. Gef1-mediated anion influx allows the establishment by the
vacuolar H.sup.+-ATPase of a proton gradient sufficient in
magnitude to drive the uphill accumulation of Na.sup.+ via
Na.sup.+/H.sup.+ exchange.
[0036] This model is entirely consistent with the physiological
data on the role of the vacuole in cation detoxification in higher
plants. As described in Example 1, to test this sequestration
model, mutant yeast strains (enal) lacking the plasma membrane
sodium efflux pump, which therefore must rely on the internal
detoxification system in order to grow on high salt, were
constructed. In theory, increasing the influx of protons into the
postulated endosomal compartment should improve Na.sup.+
sequestration via the Nhx1 exchanger. In order to increase the
H.sup.+ availability the A. thaliana gain-of-function mutant gene
AVP1-D that codes for the vacuolar pyrophosphate-energized proton
pump was expressed (FIG. 3B) (Zhen, R. G., et al., J Biol. Chem.,
272:22340-22348 (1997)). This plant pump expressed in yeast
restored the Na.sup.+ resistance of the test strain, but only if
the strain had functional NHX1 and GEF1 genes. Furthermore, Gef1p
and Nhx1p colocalize within a common organelle, the prevacuolar
compartment (Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA,
96:1480-1485 (1999)). These results strongly support the model in
FIGS. 3A and 3B and indicate that the yeast prevacuolar compartment
can be used to identify the elusive plant transporters involved
intracellular sodium detoxification.
[0037] Yeast and plant cells share pathways and signals for the
trafficking of vesicles from the Golgi network to the vacuole
(Neuhaus, J. M., et al., Plant Mol. Biol., 38:127-144 (1998);
(Paris, N., et al., Plant Physiol., 115:29-39 (1997); Sato, M. H.,
et al., J. Biol. Chem., 272:24530-24535 (1997); Vitale, A. V., et
al., Trends Plant Sci., 4:148-154 (1999)). As shown herein,
intracellular Na.sup.+ detoxification in yeast requires functional
Na.sup.+/H.sup.+ exchanger (Nhx1) and chloride channel (Gef1), and
they colocalize to a prevacuolar compartment (Gaxiola, R. A., et
al., Proc. Natl. Acad. Sci. USA, 96:1480-1485 (1999)). As described
in Example 1, to further test the utility of this system, an
Arabidopsis thaliana homologue of the yeast NHX1 gene (AtNHX1) was
cloned and its function in the nhx 1 yeast mutant was tested. The
AtNHX1 gene was able to suppress partially the cation sensitivity
phenotypes of nhx1 mutants. Further support for the role of the
Arabidopsis AtNHX1 gene in salt homeostasis came from the
observation that its expression is induced in salt-stressed plants
(Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 96:1480-1485
(1999)). A recent report shows that the overexpression of AtNHX1
gene in transgenic Arabidopsis thaliana promotes sustained growth
in soil watered with 200 mM NaCl plys 1/8 M.S. salts under
short-day cycle conditions (Apse, M., et al., Science,
285:1256-1258 (1999)). It is worth noting that every addition of
1/8 M.S. salts provides 2.5 mM potassium reducing the stringency of
the NaCl stress, and that a shortday cycle reduces oxidative
stress. As described in Example 2, transgenic plants that
overexpress the AtNHX1 were generated (35SAtNHX1 transgenics).
[0038] In plants, most of the transport processes are energized by
the primary translocation of protons. H.sup.+-translocating pumps
located at the plasma membrane and tonoplast translocated H.sup.+
from the cytosol to extracellular and vacuolar compartments,
respectively (Rea, P. A., et al., Tonoplast Adenosine Triphosphate
and inorganic Pyrophosphatase. In: Methods Plant Biochem., pp.
385-405, Academic Press Limited, London (1990)). The plant
tonoplast contains two H.sup.+-translocating pumps; the V-ATPase
and the inorganic pyrophosphatase or V-PPase. Their action results
in luminial acidification and the establishment of a H.sup.+
electrochemical potential gradient across the tonoplast (Davies, J.
M., et al., The Bioenergetics of Vacuolar H.sup.+ Pumps. In: Plant
Vacuole, pp. 340-363, Leigh, R. A., Sanders, D. (eds.), Academic
Press, San Diego (1997)). The vacuolar membrane is implicated in a
broad spectrum of physiological processes that include cytosolic pH
stasis, compartmentation of regulatory Ca.sup.2+, sequestration of
toxic ions such as Na.sup.+, turgor regulation, and nutrient
storage and retrieval. The vacuole constitute 40 to 99% of the
total intracellular volume of a mature plant cell. The vacuolar
proton pumping pyrophosphatase is a universal and abundant
component of plant tonoplast capable of generating a steady-state
transtonoplast H.sup.+ electrochemical potential similar or greater
than the one generated by the V-ATPase (Rea, P. A., et al.,
Tonoplast Adenosine Triphosphate and Inorganic Pyrophosphatase. In:
Methods Plant Biochem., pp. 385-405, Academic Press Limited, London
(1990)). Pyrophosphate (PPi) is a by-product in the activation or
polymerization steps of a wide range of biosynthetic pathways and
in plants serves as an alternative energy donor to ATP for sucrose
mobilization via sucrose synthase, for glycolysis via PPi:
fructose-6-phosphate phosphotransferase and for tonoplast
energisation via the vacuolar proton pumping pyrophosphatase
(Stitt, M., Bot. Acta 111:167-175 (1998)).
[0039] As described in Example 1, the overexpression of the A.
Thaliana gain-of-function mutant gene AVP1-D increases the
intracellular detoxification capability in yeast (Gaxiola, R. A.,
et al, Proc. Natl., Acad. Sci. USA, 96:1480-1485 (1999)). The
rationale behind this approach is that an increased influx of
H.sup.+ into the vacuolar compartment should improve Na.sup.+
sequestration via the Nhx1 exchanger. As described in Example 3, in
order to test this hypothesis in plants, a transgenic Arabidopsis
thaliana plant was engineered to overexpress the AVP1 wild-type
gene using the double tandem enhancer of the 35S promoter (Topfer,
R., et al, Nucl. Acid Res., 15:5890 (1987)). AVP1 encodes the
pyrophosphate-energized vacuolar membrane proton pump from
Arabidopsis (Zhen, R. G., et al., J. Biol. Chem., 272:22340-22348
(1997)). Previous investigations suggest that the AVP1gene is
present in a single copy in the genome of Arabidopsis (Kim, Y., et
al., Plant Physiol., 106:375-382 (1994)), however, a sequence
homologous, but not identical, to AVP1 on chromosome one has been
tentatively designated as ORF F9K20.2 on BAC F9K20 by the
Arabidopsis Genome Initiative (AGI).
[0040] Five different lines of 35SAVP1 plants showed an enhanced
salt tolerance as compared to wild-type plants in the T2 stage.
However, the most dramatic phenotype was apparent in the homozygous
T3 plants. These transgenic plants are larger than wild-type
plants. Furthermore, homozygous 35SAVP1 plants show sustained
growth in the presence of 250 mM NaCl plus 1/8 M.S. salts when
grown in a 24 hours light regimen. Interestingly, when 35SAVP1
plants were grown under short-day cycle conditions sustained growth
in the presence of 300 mM NaCl plus 1/8 M.S. salts was
observed.
[0041] Hydroponic culture increases plant growth and provides
stress-free root and shoot material (Gibeaut, D. M., et al., Plant
Physiol., 317-319 (1997)). Another important advantage of
hydrophonic culture is that we can alter the ionic composition in a
more accurate manner than in soil. These advantages could be
important for the physiological studies of salt stress. As
described in Example 4, wild type and 35SAVP1 transgenic plants
were grown hydroponically. Under such conditions the size
differences in root, leaves and stems among wild type and 35SAVP1
transgenic plants are dramatic. To learn about the salt tolerance
of these plants under hydroponic conditions, NaCl concentration
were increased stepwise by 50 mM every 4 days (Apse, M., et al.,
Science, 285:1256-1258 (1999)). 35SAVP1 transgenic plants appear
healthy in the presence of 200 mM NaCl while wild type controls
show severe deleterious effects in their leaves and stems.
[0042] Genetic engineering promises to transform modem agriculture.
Salinization of soil due to irrigation has rendered much land
unusable for crop production. Described herein is a strategy using
genetic and molecular biological approaches to improve the
intracellular Na.sup.+ detoxification capabilities of crops. The
fact that genetically engineered Arabidopsis thaliana plants that
overexpress either AVP1 (the pyrophosphate-energized vacuolar
membrane proton pump, this work) or AtNHX1 (the Na.sup.+/H.sup.+
antiporter, (Apse, M., et al., Science, 285:1256-1258 (1999)) and
this work) are capable of growing in the presence of 200 mM NaCl
strongly supports the strategy described herein. It is likely that
a double transgenic plant will show a further enhanced
salt-tolerant phenotype. Furthermore, it is expected that these
Arabidopsis thaliana transporters or their counterparts will be
able to perform similar function in important agricultural crops.
The increased size of 35SAVP1 Arabidopsis transgenic plants also
contribute to future food security, namely potential yield
increases in genetically engineered crops.
EXEMPLIFICATION
Example 1
The Arabidopsis Thaliana Proton Transporters, AtNhx1 and Avp1, can
Function in Cation Detoxification in Yeast
Materials and Methods
[0043] Yeast strains and Plasmids. All strains used are isogenic to
W303 (ura3-1 can1-100 leu2-3, 112trp1-1 his3-11, (Gaxiola, R. A.,
et al, EMBO J., 11:3157-3164 (1992)). Plasmids pRG52 (Agef1::HIS3)
(Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050
(1998)) and pRG197 (.DELTA.nhx1::HIS3) were used to construct the
deletions of GEF1 and NHX1 genes, yielding strains RGY85 and
RGY296, respectively. The ena1::HIS3 mutant was obtained from Fink
Lab collection (L5709). Transformation was performed by using the
lithium acetate method (Gietz, D., et al., Nucleic Acids Res.,
20:1425 (1992)). Double mutants RGY324 (gef1::HIS3 ena1::HIS3),
RGY326 (nhx1::HIS3 ena1::HIS3), and RGY343 (gef1::HIS3 nhx1::HIS3)
were obtained by crossing the single-mutant strains. Double mutants
were identified among the meiotic progeny by scoring for the
phenotypes associated with each of the single mutants. Sporulation,
tetrad dissection, and mating types were scored as described
(Guthrie C. and Fink, G. R., Guide to Yeast Genetics and Molecular
Biology (Academic, San Diego (1991)). Cells were grown in YPD (1%
yeast/2%/opeptone/2% dextrose; Difco), YPGAL (1%
yeast/2%/opeptone/2% galactose; Difco), SD (Difco; Synthetic medium
with 2% Dextrose), or APG (APG is a synthetic minimal medium
containing 10 mM arginine, 8 mM phosphoric acid, 2% glucose, 2 mM
MgSO.sub.4, 1 mM KCl, 0.2 mM CaCl.sub.2, and trace minerals and
vitamins) (Rodriguez-Navarro, A. and Ramos, J., J Bacteriol.,
159:940-945 (1984)). MnCl.sub.2 (Sigma), tetramethylammonium
chloride (Sigma), NaCl (Sigma), or hygromycin-B (Sigma were added
as indicated.
[0044] Wild type, L5709 (ena1:.HIS3), RGY324 (gef1::HIS3
ena1:.HIS3), and RGY326 (nhx1::HIS3 ena1::HIS3) strains were
transformed with pYES2 vector (Invitrogen) and plasmid
pYES2-AVP1-E229D described in ref. Zhen, R. G., et al., J. Biol.
Chem., 272:22340-22348 (1997). The strain RGY343 (gef1::HIS3
nhx1::HIS3), used for histochemical analysis, was transformed with
pRG151 (GEF1-GFP) (Gaxiola, R. A., et al. Proc. Natl. Acad. Sci.
USA, 95:4046-4050 (1998)) and with pRIN73 [NHX1-(HA).sub.3] (Nass,
R., and Rao, R., J. Biol. Chem., 273:21054-21060 (1998)).
[0045] Wild-type and RGY296 (nhx1::HIS3) strains were transformed
with vector pAD4 (Ballester, R., et al., Cell, 59:681-686 (1989)).
RGY296 (nhx1::HIS3) was transformed with pRG308 (ADH1::AtNHX1) (see
Cloning of AtNHX1).
[0046] Determination of Intracellular Sodium and Potassium content.
Cells were grown overnight in SD-ura medium (Difco; synthetic
medium with 2% dextrose without uracil). YPGAL (1% yeast extract/2%
peptone/2% galactose; Difco) media was inoculated with the
overnight stocks and grow to an A.sub.600 of 0.6. At this OD, NaCl
was added to a final concentration of 0.7 M. The cells incubated
for 6 h, harvested by centrifugation, washed two times with 1.1 M
sorbitol and 20 mM MgCl.sub.2, and entracted with water for 30 min
at 95.degree. C. The amount of Na.sup.+ and K.sup.+ in cells was
determined at the University of Georgia Chemical Analysis
Laboratory by an Inductively Coupled Plasma-MS (see
http://www.rserv/uga.edu/rsnew/chemicalanalysis/). Intracellular
cation concentrations were estimated as described (Gaxiola, R. A.,
et al., EMBO J., 11:3157-3164 (1992)) by using the intracellular
water value calculated for cells grown in 1M NaCl.
[0047] Immunofluorescence. The strain RGY343 (gef1::HIS3
nhx1::HIS3) was grown in SD-ura, -leu medium (Difco; synthetic
medium with 2% dextrose without uracil and leucin) to
mid-logarithmic phase, 0.1 mg/ml hygromycin B was added, and the
culture was incubated for 1 h at 30.degree. C. Cells were fixed
with 3.7% formaldehyde (Sigma) for 45 min at room temperature
without agitation. Spheroplast formation, permeablization, washing,
and antibody incubation was performed as described (Pringle, J., et
al., in Immunofluorescence Methods for Yeast, eds. Guthrie, C. And
Fink, G. F. (Academic, Sand Diego), Vol. 194 pp.565-602 (1991)).
MAB HA11 used as first antibody was from Babco (Richmond, Calif.).
Cy3-conjugated goat antimouse IgG was from Jackson Immunoresearch.
4',6-Diamidino-2-phenylind- ole (Sigma) was added to mounting
medium to stain mitochondrial and nuclear DNA.
[0048] Subcellular Fractionation and Western Analysis. The strain
RGY343 (gef1::HIS3 nhx1::HIS3) was grown in APG medium (pH 7.0),
and lysates fractioned on a 10-step sucrose density gradient as
described (Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060
(1998)). Aliquots of individual fractions (100 .mu.g) were
subjected to SDS/PAGE and transferred to nitrocellulose as
described (Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060
(1998)). Western blots were probed with monoclonal anti-GFP (green
fluorescent protein) antibody (1:10,000 dilution; CLONTECH),
anti-hemagglutinin antibody (1:10,000 dilution: Boehringer
Mannheim), and peroxidase coupled goat anti-mouse antibody
(1:5,000;) and developed by using the ECL enhanced
chemiluminescence system (Amersham Pharmacia).
[0049] Plant Strains, Growth conditions and RNA Preparation. A.
thaliana plants (ecotype Columbia) were grown aseptically on
unsupplemented plant nutrient agar without sucrose (Haughn, G. W.
and Somerville, C., Mol. Gen. Genet., 204:430-434 (1986)) for 15
days at 19.degree. C. and under continuous illumination. NaCl or
KCl was added to a final concentration of 250 mM, and the plants
were incubated for 6 h. Total RNA from tissue of salt-treated and
untreated plants was isolated (Niyogi, K.K. and Fink, G. R., Plant
Cell, 4:721-733 (1992)), Hybond-N (Amersham) membranes were
hybridized with a .sup.32P-Labeled DNA probe from plasmid pRG308.
Hybridization was performed at 65.degree. C. overnight. Washes were
performed at 65.degree. C. with 0.2% standard saline citrate
(SSC)/0.1% SDS (Ausebel, F., et al, Curr. Protocols in Mol Biol.
(Wiley, N.Y.) (1988)). 18S probe was used as loading control
(Unfried, I., et al., Nucleic Acids Res., 17:7513 (1989)). MACBAS
2.4 program was used to quantify the relative amount of RNA.
[0050] Cloning of AtNHX1. AtNHX1 was cloned from a phage cDNA
library of A. thaliana (Kieber, J. J., et al., Cell, 72:427-441
(1993)) (obtained from the Arabidopsis Biological Resource Center)
by probing with an expressed sequence tag (Arabidopsis Biological
Resources Center, DNA Stock Center) containing a partial clone. A
full-length clone (2.1 kB) was ligated into vector pSK2
(Stratagene) at the NotI sit, generating plasmid pRG293. The AtNHX1
ORF was amplified via PCR by using pRG293 as template and
GGCCCGGGATGGATTCTCTAGTGTCGAAACTGCCTTCG (SEQ ID NO: 5) (italicized
bases correspond to nucleotides 1-30 of the ORF) and T7
oligonucleotides. The PCR product was then digested with XbaI and
SalI and ligated into pAD4 vector generating plasmid pRG308. The
AtNHX1 ORF was sequenced to verify the fidelity of the PCR product.
The full-length sequence is longer than the ORF reported by the
Arabidopsis Genome Initiative (A TM021B04.4), and has been
deposited in GenBank (accession no. AF106324).
Results
[0051] The Arabidopsis Vacuolar H.sup.+-Pyrophosphatase (Avp1)
Confers Salt Tolerance to Yeast enal Mutants. To determine the
components of the intracellular system required for sodium
detoxification, an enal mutant that lacks the plasma membrane
sodium efflux pump and therefore must rely on the internal
detoxification system to overcome sodium toxicity was used. Growth
of the enal strain is sensitive to low concentrations of sodium
(200 mM), concentrations that do not inhibit the growth of
wild-type strains. The sequestration model (Nass, R. and Rao, R.,
J. Biol. Chem., 273:21054-21060 (1998) and, Gaxiola, R. A., et al,
Proc. Natl. Acad. Sci. USA, 95:4046-4050 (1998)) predicts that the
enal strain would become salt tolerant if one could enhance the
availability of protons in the postulated endosomal compartment.
With increased influx of protons, cytoplasmic Na.sup.+ would be
sequestered via the Nhx1 exchanger. The yeast vacuolar ATPase is a
multisubunit protein, so it is difficult to increase its activity
by overexpressing any one of its subunits. However, it is possible
to increase the influx of protons by expressing the A. thaliana
AVP1 gene in yeast. This gene encodes a single polypeptide that,
when expressed in yeast, is capable of pumping protons into the
lumen of the vacuole (Kim, E. J., et al., Proc. Natl. Acad. Sci.
USA, 91:6128-6132 (1994)). To ensure maximum activity of this
proton pump, the E229D gain-of-function mutant of the AVP1 gene
(AVP1-D) that has enhanced H.sup.+ pumping capability was expressed
(Zhen, R. G., et al., J. Biol. Chem., 272:22340-22348 (1997)).
[0052] Overexpression of AVP1-D restored salt tolerance to
salt-sensitive enal mutants. The restoration of salt tolerance to
an enal strain by AVP1-D requires functional NHX1 and GEF1 genes:
enalnhx1 AVP1-D and enal gef1 AVP1-D strains are salt
sensitive.
[0053] Expression of Arabidopsis vacuolar pyrophosphatase AVP1 in
enal mutants: Vector pYES2 (Invitrogen) was introduced into
wild-type, ena1, ena1 nhx1, and ena1 gef1 mutants. Plasmid
pYes2-AVP1-D (Zhen, R. G., et al., J. Biol. Chem., 272:22340-22348
(1997)) was introduced into enal, enal nhx1, and ena1 gef1 mutants.
Five-fold serial dilutions (starting at 10.sup.5 cells) of each
strain were plated on YPGAL (1% yeast extract/2% peptone/2%
galactose) with or without 0.5 M NaCl and incubated at 30.degree.
C. for 2 days. FIGS. 1A and 1B show intracellular concentrations of
Na.sup.+ and K.sup.+. Exponentially growing cells (wild-type and
enal transformed with pYES2 vector and ena1, ena1 nhx1, and enal
gef1mutants carrying pYes2-AVP1-D) were exposed to 0.7M NaCl or 6
hours. Total cell extracts were prepared (see Materials and
Methods), and Na.sup.+ and K.sup.+ concentrations were determined.
There is a consistent reduction in total cell Na.sup.+ in the enal
AVP-D strain. The reason for this reduction is unknown.
[0054] The intracellular Na.sup.+ and K.sup.+ contents of wild-type
strains and of strains carrying various mutations affecting sodium
tolerance were determined after 6 h of exposure to media
supplemented with 0.7 M NaCl (FIGS. 1A and 1B). The intracellular
Na.sup.+ content in the enal mutant is 8-fold higher than in the
wild-type strain. The enal AVPD strain is salt-resistant, even
though its intracellular Na.sup.+ content is 4-fold higher than
that of the wild type. In enal AVP1-D strains lacking either gef1
or nhx1 (i.e., enal gef1 or enal nhx1), the Na.sup.+ content is not
reduced to the extent that it is in GEF1 NHX1 strain. Taken
together, the genetic and physiological data are consistent with
the model that Nhx1, Gef1 and Avp1 cooperate to sequester sodium
internally.
[0055] The intracellular K.sup.+ content correlates with salt
tolerance and is inversely correlated with the Na.sup.+ content of
our strains (FIG. 1B). The wild-type K.sup.+ concentration is
.apprxeq.100 mM but is reduced to 20 mM in the enal mutant.
Interestingly, in an enal strain that overexpresses the AVP1-D
gene, the intracellular concentration of K.sup.+ is restored almost
to wild-type levels (FIG. 1B). However, AVP1-D overexpression fails
to restore wild-type levels of intracellular potassium unless both
NHx1 and GEF1 are functional (see the double mutants enal nhx1 or
enal gef1 in FIG. 1B).
[0056] The NHX1 and GEF1 genes, which have been identified as
important in sodium detoxification, are also required for the
detoxification of other cations. Growth of gef1 and nhx1 mutants in
the presence of toxic cations: Five-fold serial dilutions (starting
at 10.sup.5 cells) of the indicated strains were grown at
30.degree. C. for 2 days on YPD (1% yeast extract/2% peptone/2%
dextrose) with the addition of either 3 mM MnCl.sub.2, 0.45 M
tetramethylammonium (TMA), or 0.05 mg/ml hygromycin B (HYG) as
indicated.
[0057] For example, gef1 mutants are sensitive to 3 mM MnCl.sub.2,
0.45 M tetramethylammonium chloride and to 0.05 .mu.g/ml
hygromycin-B. The nhx1 mutant is also sensitive to
tetramethylammonium chloride and hygromycin. The extreme
sensitivity of the nhx1 mutant to hygromycin provides an important
tool for assaying nhx1 function.
[0058] Gef1p and Nhx1p Colocalize. The sequestration model
postulates not only a functional connection between the anion
channel Gef1 and sodium exchanger Nhx1 but also predicts that these
two proteins colocalize within a common compartment. Because
previous studies indicated that Nhx1 localizes to a prevacuolar
compartment (Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060
(1998)), two types of experiments were performed to determine
whether Gef1 and Nhx1 proteins colocalize to this compartment.
[0059] Distribution of fluorescence and immunodetection of
subcellular fractions in gef1 nhx1 cells transformed with two
constructs: a GEF1-GFP fusion and a NHX1-(HA).sub.3-tagged fusion
were determined. The strain RGY419 (gef1 nhx1) was transformed with
plasmids pRG151; GEF1-GFP and pRIN73; NHX1-(HA).sub.3.
Transformants were grown in SD (Difco; synthetic medium with 2%
dextrose). When the cells reached OD.sub.600=0.5, hygromycin B
(Sigma) was added to a final concentration of 0.1 mg/ml and the
cells were incubated for 40 min at 30.degree. C. Cells were fixed
and stained with antibodies to HA epitope and
4',6-diamidino-2-phenylindole (DAPI). Cells were viewed by
charge-coupled device microscopy and optically sectioned by using a
deconvolution algorithm (Scanalytics, Billerica, Mass.) (Kennedy,
B. K., et al, Cell, 89:381-391 (1997)); (Bar=1 .mu.m.).
[0060] It was found that hemagglutinin (HA)-tagged Nhx1 and
Gef1-GFP fusion protein colocalize as shown via epifluorescence
deconvolution microscopy (FIG. 3A). Persistence of signal
coincidence on 90.degree. rotation of the image further supports
colocalization of the two transporter proteins in these cells.
[0061] The colocalization of Nhx1 (HA).sub.3 and GEF1-GFP is also
supported by the comigration of the two proteins in sucrose density
gradients of membrane preparations obtained from cells expressing
the tagged proteins. The strain RGY419 (gef1 nhx1) transformed with
plasmids pRG151; GEF1-GFP and pRIN73; NHX1-(HA).sub.3 was grown in
APG medium (Rodriguez-Navarro, A. and Rea, P. A., J. Biol. Chem.,
159:940-945 (1984)), converted to spheroplasts, lysed, and
fractionated on a 10-step sucrose gradient (18-54%) as described
(Sorin, A., et al., J. Biol. Chem., 272:9895-9901 (1997) and
Antebi, A. and Fink, G. R., Mol. Biol. Cell, 3:633-654 (1992)).
Western blots showed the distribution of Gefl-GFP and Nhx1-HA (see
Example 1, Materials and Methods).
[0062] The sedimentation behavior of the membrane fraction
containing both proteins is consistent with that of a prevacuolar
compartment (Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060
(1998)). Gef1-GFP (but not Nhx1) is also present in Golgi
fractions, consistent with previous studies (Gaxiola, R. A., et
al., Proc. Natl. Acad. Sci. USA, 95:4046-4050 (1998), Schwappach,
B., et al., J. Biol. Chem., 273:15110-15118 (1998)).
[0063] An A. thaliana Homologue of NHX1 Functions in Yeast. The
yeast strain described herein provides an important tool for
identifying genes that mediate salt tolerance in other organisms.
To test the utility of this system, a sequence from Arabidopsis
(See Materials and Methods) with very high homology to the S.
cerevisiae NHX1 ORF was identified and used an expressed sequence
tag (see Materials and Methods) to obtain a full-length clone of
this Arabidopsis gene. An alignment of the amino acid sequences of
Nhx1 homologues from Arabidopsis (AtNhx1), human (HsNhe6), and
yeast (ScNhx1) reveals segments of amino acid identity and
similarity within predicted transmembrane domains (FIG. 2).
However, it is important to note that despite these relationships,
neither the--nor the C-terminal regions of AtNhx1 and ScNhx1 show a
high degree of homology (FIG. 2). A characteristic of mammalian
Na.sup.+/H.sup.+ antiporters is their inhibition by amiloride. A
putative amiloride binding site (.sup.163DVFFLFLLPPI.sup.173) (SEQ
ID NO: 4) has been defmed via point mutants in the human NHE1
antiporter gene (Counillon, L., et al., Proc. Natl. Acad. Sci. USA,
90:4508-4512 (1993)). AtNhx1, HsNhe-6 and ScNhx1 have an almost
identical sequence (FIG. 2). However, our attempts to inhibit the
activity of either Nhx1 or AtNhx1 in yeast cultures with amiloride
were unsuccessful.
[0064] The extreme sensitivity of yeast nhx1 mutants to hygromycin
permitted the testing of whether the cloned Arabidopsis AtNHX1 ORF
could provide Na.sup.+/H.sup.+ exchange function in yeast. Vector
pAD4 (Ballester, R., et al., Cell, 59:681-686 (1989) was introduced
into wild-type and nhx1 strains. Plasmid pRG308; ADH; AtNHX1 was
introduced into nhx1 mutants as indicated. Five-fold serial
dilutions (starting at 10.sup.5 cells) of the indicated strains
were grown at 30.degree. C. for 2 days on YPD (-) or on YPD
supplemented with 0.05 mg/ml hygromycin (+). Serial dilutions of
the same strains were grown on APG medium (see Materials and
Methods) (-) or on APG supplemented with 0.4 M NaCl
(Rodriguez-Navarro, A. and Ramos, J., J. Bacteriol., 159:940-945
(1984).
[0065] The At NHX1 gene is capable of suppressing the hygromycin
sensitivity of the nhx1 mutant. The AtNHX1 gene also suppressed the
NaCl sensitivity of nhx1 mutant but only under conditions in which
the K.sup.+ availability was reduced. However, AtNHX1 was not
capable of rescuing the Na.sup.+- sensitive growth phenotype of the
double mutant enal nhx1 overexpressing the AVP1-D gene.
[0066] Further support for the role of the Arabidopsis AtNHX1gene
in salt homeostasis came from an analysis of its expression in
salt-stressed plants. Plants were grown for 15 days under standard
conditions and then exposed for 6 h to either 250 m NaCl or KCl.
The NaCl stress increased AtNHX1 mRNA levels 4.2-fold, whereas KCl
promoted only a 2.8-fold increase. This increase in mRNA level
produced by sodium resembles that described for the yeast NHX1 gene
(Nass, R. and Rao, R., J. Biol. Chem., 273:21054-21060 (1998)). RNA
tissue blot hybridized with AtNHX1. Ten micrograms of total RNA
from 15-day old plants exposed to 250 mM NaCl or KCl for 6 h and a
control grown without salt was subjected to electrophoresis on a
denaturing formaldehyde gel. The blot was hybridized with a probe
internal to AtNHX1 ORF. An 18S ribosomal probe was used as a
loading control.
Discussion
[0067] The studies described herein provide evidence for the
importance of the prevacuolar pH for intracellular Na.sup.+
sequestration in yeast. Overexpression of the plant
H.sup.+-pyrophosphatase (Avp1) confers salt tolerance to yeast only
in those strains containing a functional chloride channel (Gefl)
and the Na.sup.+/H.sup.+ exchanger (Nhx1).
[0068] These data support a model in which the Nhx1
Na.sup.+/H.sup.+ exchanger acts in concert with the vacuolar ATPase
and the GEF1 anion channel to sequester cations in a prevacuolar
compartment. Several studies suggest that the prevacuolar
compartment may be derived both from the plasma membrane and the
late Golgi. These vesicles are likely involved in the assembly of
the vacuole or delivery of cargo to this organelle. It is
reasonable to expect that these prevacuolar vesicles detoxify
cations by sequestration, thereby lowering their concentrations in
the cytoplasm and in other organelles.
[0069] The yeast system described herein permits the functional
assessment of diverse heterologous proteins in salt tolerance:
chloride channels, H.sup.+ pumps, and Na.sup.+/H.sup.+ exchangers
and other cation/H.sup.+ exchnagers or cation/bicarbonate
symporters. The system is robust and flexible. The function of the
Arabidopsis chloride channels (Gaxiola, R. A., et al., Proc. Natl.
Acad. Sci. USA, 95:4046-4050 (1998), Hechenberger, M., et al., J.
Biol. Chem., 271:33632-33638 (1996)), H.sup.+ pump, and
Na.sup.+/H.sup.+ exchanger can be assayed in the corresponding
yeast mutant. Despite the inability of At NHX1 to suppress all the
phenotypes of the yeast nhx1 mutant, the fact that it suppresses
some phenotypes, coupled with the DNA homology between AtNHX1 and
yeast NHX1, indicates that the plant gene carries out a similar
function to that of the yeast homologue. The observation that the
AtNHX1 l gene suppresses the sensitivity of the nhx1 mutant to
hygromycin but provides only a weak Na.sup.+ detoxification
phenotype could be a consequence either of differential regulation
of the transporters in the two organisms or of distinct cation
transport selectivities.
[0070] The regulation of AtNHX1 by salt and the ability of the
plant gene to suppress the yeast nhx1 mutant suggest that the
mechanism by which cations are detoxified in yeast and plants may
be similar. Indeed, previous work suggested that vacuolar sodium
accumulation in salt-tolerant plants may be mediated by a tonoplast
Na.sup.+/H.sup.+ antiporter that utilizes the proton-motive force
generated by the vacuolar H.sup.+-ATPase (V-ATPase) and/or
H.sup.+-translocating pyrophosphatase (V-PPase; refs. Barkla, B.
J., et al., Symp. Soc. Exp. Biol., 48:141-153 (1994), Zhen, R. G.,
et al., The Molecular and Biochemical Basis of
Pyrophosphate-Energized Proton Translocation at the Vacuolar
Membrane (Academic, San Diego), Kirsh, M, et al., Plant Mol. Biol.,
32:543-547 (1996)).
[0071] The finding described herein that both gef1 and nhx1 mutants
are hypersensitive to hygromycin indicate that the level of
resistance to hygromycin depends on the function of the vacuolar
and prevacuolar organelles. Yeast mutants impaired in K.sup.+
uptake (trk1) are hypersensitive to hygromycin (Madrid, R., et al.,
J. Biol. Chem., 273:14838-14844 (1998)); reduced K.sup.+ uptake
hyperpolarizes the plasma membrane potential and drives the uptake
of alkali cations such as hygromycin. Mutations that reduce the
H.sup.+ pumping activity of the plasma membrane H.sup.+-ATPase,
Pmal, depolarize the plasma membrane potential and confer
resistance to hygromycin (McCusker, J. H., et al., Mol. Cell.
Biol., 7:4082-4088 (1987)). Thus, mutants such as gef1 or nhx1 that
affect the pH or membrane potential of the vacuolar and prevacuolar
compartments may be expected to affect hygromycin
compartmentation.
EXAMPLE 2
Transgenic Plants that Overexpress the AtNHX1
[0072] Transgenic plants that overexpress the AtNHX1 were generated
using Agrobacterium-mediated plant transformation. The transgenic
AtNHX1 was expressed using a double tandem enhancer of the 35S
promoter of CaMV (Topfer, R., et al., Nucl. Acid Res., 15:5890
(1987)). T3 transgenic plants are less affected than wild type
controls when watered with 300 mM NaCl.
[0073] 15 wild-type plants and 15 35SAtNHX1 transgenic were grown
on a 12 hours-day cycle for 20 days. During this period plants were
watered every 5 days with a diluted nutrient solution (1/8 M.S.
salts). 200 mM NaCl was added to the watering solution at day 21
and at day 33 plants were watered with a nutrient solution
containing 300 mM NaCl. Plants were photographed 10 days after the
last NaCl treatment.
EXAMPLE 3
Salt-stressed Wild Type Plants and 35SAVP1 Transgenics
[0074] Transgenic plants that overexpress AVP1 were generated using
Agrobacterium-mediated plant transformation. The transgenic AVP1
was expressed using a double tandem enhancer of the 35S promoter of
CaMV (Topfer, R., et al., Nucl. Acid Res., 15:5890(1987)). 15
wild-type plants and 15 35SAVP1 transgenics were grown on a 24
hours-day cycle for 16 days. During this period plants were watered
every 4 days with a diluted nutrient solution (1/8 M.S. salts). 200
mM NaCl was added to the watering solution at day 17 and at day 27
plants were watered with nutrient solution containing 250 mM NaCl.
Plants were photographed 10 days after the last NaCl treatment.
Identical conditions and treatment as described in Example 2 were
used.
[0075] These transgenic plants are larger than wild-type plants.
Furthermore, homozygous 35SAVP1 plants show sustained growth in the
presence of 250 mM NaCl plus 1/8 M.S. salts when grown in a 24
hours light regimen. Interestingly, when 35SAVP1 plants were grown
under short-day cycle conditions (12 hour day/light cycle)
sustained growth in the presence of 300 mM NaCl plus 1/8 M.S. salts
was observed.
EXAMPLE 4
Hydroponically Grown Wild Type and 35SAVP1 Transgenic Plants
[0076] Hydroponically grown wild type and 35SAVP1 transgenic plants
were generated. 65 days old wild type and 35SAVP1 transgenic plants
grown in solution culture on a 12 hour light cycle.
[0077] Wild type and 35SAVP1 transgenic plants were also grown in
solution culture on a 12 hours light cycle for 20 days. Starting at
day 21, NaCl concentration was increased in a stepwise fashion by
50 mM increments every 4 days. Plants were photographed after 4
days in the presence of 200 mM NaCl.
EXAMPLE 5
Double Transgenic Plant with 35SAVP1 and 35SAtNHX1
[0078] Overexpression of the pyrophosphate-energized vacuolar
membrane proton pump AVP1 likely increases the availability of
H.sup.+ in the lumen of the vacuole, and the AtNHX1
Na.sup.+/H.sup.+ antiporter uses these H.sup.+ to sequester
Na.sup.+ cations into the vacuole. Therefore, higher expression of
these transporters likely maximizes the sequestration capability of
the vacuole. To generate transgenic Arabidopsis plants that
overexpress both genes AVP1 and AtNHX1, T3 35S AVP1 plants are used
as females and T3 35S AtNHX1 plants are used as males. Female
plants are hand-emasculated and anthers from freshly opened flowers
of donor plants are harvested. With these anthers the emasculated
plants are pollinated by touching the anthers onto the stigmas. The
pollinated flowers are labeled and any remaining opened or unopened
flowers from the same female plant are removed to avoid any
confusion at harvest. The harvested seeds are sterilized using a
50% sodium hypochloride solution and mixed vigorously for 5 minutes
and rinsed with water thoroughly. The sterilized seeds are stored
in soft agar over night at 4.degree. C. Then they are sprinkled
onto solidified kanamycin-hygromycin selective medium. The 35S AVP1
construct has the neomycin phosphotransferase II gene that confers
kanamycin tolerance in plants while the 35SAtNHX1 construct has a
modified hygromycin B phosphotransferase that confers hygromycin
tolerance in plants. The resistant seedlings are transplanted into
soil and to the hydroponic media to be tested for their
salt-tolerant phenotype. A transgenic Arabidopsis thaliana plant to
overexpress the A. thaliana gain-of-function mutant gene AVP1-D
(Zhen, et al., J. Biol. Chem., 272:22340-22348 (1997)) is
engineered using the same double tandem enhancer of the 35A
promoter described above (Topfer, R., et al., Nuc. Acid Res.,
15:5890 (1997)). Plants overexpressing the gain of function mutant
gene will likely show an enhanced phenotype. These plants are
characterized in parallel with the 35SAVP1, 35S AtNHX singles and
doubles trangenics. The A. thaliana gain-of-function mutant gene
AVP1-D is subcloned into plasmid pRT103 carrying the 35S promoter
and the polyadenylation signal of CaMV (Topfer, R., et al., Nucl
Acid Res., 15:5890 (1997)). A HindIII fragment containing the
chimeric 35SAVP-D gene is subcloned into pBIBhyg (Becker, D., Nucl
Acid Res., 18:203 (1990)). The resulting T-DNA vector is
transformed into Agrobacterium tumefaciens strain GV3101 via
electroporation, and used for subsequent vacuum infiltration of
Arabidopsis thaliana ecotype Columbia (Bechtold, N., et al., C.R.
Jeances Acad. Sci. Ser. III Sci. Vie, 316:1194-1199 (1993)).
Integration is confirmed on Southern blots of T3 plants and
expression monitored on Northern blots of positive T3 plants.
EXAMPLE 6
Comparative Transport Study With Vacuoles From The Roots Of
Wild-Type and 35S AVP1 Transgenic Plants
[0079] The purpose of this study is to determine if the vacuoles of
35SAVP1 transgenic plants show a higher proton transport activity
dependent on pyrophosphate. These determinations are done with root
and shoot tissues separately from plants grown hydroponically. The
transgene could show a tissue-specific regulation despite the 35S
promoter.
[0080] In order to compare the PPI-dependent H.sup.+ translocation
activities of wild-type and 35S AVP1 transgenic plants sealed
tonoplast-enriched vesicles from roots and leaves of the above
plants are prepared. The homogenization and differential
centrifugation procedure described by Rea and Turner (Rea, P. A.,
et al., Tonoplast Adenosine Triphosphate and Inorganic
Pyrophosphatase. In: Meth. Plant Biochem., pp. 385-405, Academic
Press limited, London (1990)) is followed. H.sup.+ translocation is
assayed fluorimetrically using acridine orange (2.5 .mu.M) as
transmembrane pH difference indicator in assay media containing
vacuole membrane-enriched vesicles as described by Rea and
coworkers (Zhen, R. G., et al., J. Biol. Chem., 272:22340-22348
(1997)). The assay media contains 300 .mu.M Tris-PPi, 50 mK KC1,
2.5 .mu.M acridine orange, 5 mM Tris-Mes (pH 8.0). Intravesicular
acidification is triggered with the addition of 1.3 mM MgSO4 and
terminated with the addition of the protonophore FCCP at 2.5 .mu.M.
Fluorescence is measured at excitation emission wavelengths of 495
and 540 nM, respectively, at a slit width of 5 nM (Zhen, R. G., et
al., J. Biol. Chem., 269:23342-23350 (1994)). A further test to
support that the H.sup.+ translocation is AVP1 driven is the
addition of the specific inhibitor aminomethyledediphosphonate
(Zhen, R.G., et al., Plant Physiol., 104:153-159 (1994)).
EXAMPLE 7
Determination Of The Na.sup.+/K.sup.+ Ratios In Leaves And Stems Of
The Transgenic Plants
[0081] These measurements indicate to whether or not the transgenic
plants described herein have an increased vacuolar capacity to
sequester Na.sup.+ in their leaves cells or elsewhere. Toxic
concentrations of NaCl build up first in the fully expanded leaves
where NaCl is compartmentalized in the vacuoles. Exposure to NaCl
can disrupt or reduce K.sup.+ uptake leading to K.sup.+ deficiency
and growth inhibition (Wu, S.J., et al., Plant Cell, 8:617-627
(1996). A cytosolic consequence of reduced K.sup.+ content and high
Na.sup.+ is the inhibition of important enzymes. An example of such
enzymes is the 3'(2'), 5'-bisphosphate nucleotidase of yeast whose
activity is more sensitive to Na.sup.+ when K.sup.30 content is low
(Murguia, J. R., et al., Science, 267:232-234 (1995). To determine
the Na.sup.+/K.sup.+ ratios in leaves and stemS wild-type and
35SAVP1/35SAtNHX1 double and single transgenics in hydroponic
conditions (Gibeaut, D. M., et al., Plant Physiol., 317-319 (1997)
are grown. NaCl is added to the growth media in a stepwise fashion
starting with 50 mM up to 250 mM (Apse, M., et al., Science,
285:1256-1258 (199). At every point the rosette and the stems of
the treated plants are collected and their weight is determined.
The samples are dried out in an oven at 80.degree. C. and their dry
weight is determined. The dry samples are boiled in a determined
volume of water and their Na.sup.+ and K.sup.+ contents determined
via atomic absorption spectrophotometry (Apse, M., et al., Science,
285:1256-1258 (1999); Gaxiola, R., et al., Embo J., 11:3157-2164
(1992)).
EXAMPLE 8
Determination Of Whether 35S AVP1 Transgenic Plants Are Larger
Because Their Cells Are Larger Or Because They Have More Cells, Or
Both
[0082] The shoot meristems labeling index is compared with one of
the wild-type plants. Morphological and anatomical observations
measuring and counting cells of leaves, roots and stems are
performed. To determine if 35S AVP1 transgenic plants are larger
because they have more cells, their shoot meristems labeling index
is compared with the one of wild-type plants. To measure the DNA
synthesis or cell proliferation 5-Bromo-2'-deoxy-uridine (BrdU)
that can be incorporated into DNA in place of thymidine is used.
Cells that have incorporated BrdU into DNA are detected using a
monoclonal antibody against BrdU monoclonal antibody and an
anti-mouse Ig-alkaline phosphatase as a second antibody. The bound
anti-BrdU monoclonal antibody is visualized by light microscopy and
the ratio between DAPI stained and BrdU positives established. The
protocol is a modification of the one published by Chiatante and
coworkers (Levi, M., et al., Physiol. Plant. 71:68-72 (1987)) and
the BrdU labeling and detection kit II from Boehringer Mannheim.
The plants are exposed for different times to the BrdU labeling
medium and then fixation, paraffin embedding and sectioning is
performed as described by Meyerowtz and coworkers (Drews, G., et
al., Plant Mol. Biol. Rep., 5:242-250 (1988)). For observation of
leaf tissue, fresh tissues are embedded in 5% agarose and slice
them with a microslicer. For primary root observation, seedlings
are fixed for 4 hr in 50% ethanol, 5% acetic acid, and 3.7%
formaldehyde at room temperature, dehydrate them in graded ethanol
series, permeate them with xylene, and infiltrate them with
paraffin. Eight-micrometer sections are stained with 0.05%
toluidine blue and cells are counted under a microscope. As an
alternative for the visualization and determination of cell size
the method described by Greenberg and coworkers (Rate, et al., The
Plant Cell, 11:1695-1708 (1999)) is followed.
EXAMPLE 9
Isolation Of Mutants In The Transporters
[0083] Genetic approaches are very powerful in analyzing complex
biological traits (Serrano, R., Crit. Rev. Plant Sci., 13:121-138
(1994)) Reverse genetics is a very important new tool for plant
biologists. The generation of a good collection of tagged knockouts
by Sussman and coworkers (Krysan, P., et al., Proc. Natl. Acad.
Sci. USA, 93:8145-8150 (1996)) has open a very important avenue for
the analysis of gene disruptions in Arabidopsis. the Arabidopsis
Knock-out Facility of the University of Wisconsin Madison
(http://www.biotech.wisc.edu/NewServicesA- ndResearch/Arabidopsis)
is used to search among the 60,480 Arabidopsis (ecotype WS) lines
that have been transformed with the T-DNA vector pD991 for the
presence of T-DNA inserts within AtCLC-c, AtCLC-d, AVP1, AtNHX1 and
their homologues. The phenotypes of the above knock-outs will shed
light towards the understanding of the physiological roles of these
transporters in normal and stress conditions. An initial
characterization of the knockout plants includes testing for their
salt tolerance and their Na.sup.+/K.sup.+ ratios. The generation of
double knock-outs via crosses help to further understand the
interaction among the transporters as well as the crosses with the
35SAVP1 and the 35SAtNHX1 transgenic plants.
[0084] To search for Arabidopsis knock-out PCR primers are designed
following the guidelines detailed in the University of Wisconsin
web site. Tested primers are sent to UW-Madison, where 62 PCR
reactions that are sent to us for Southern blot analysis are
performed. Positive PCR products are sequenced. If the sequence
reveals that there is a T-DNA inserted within the gene the gene
specific primers are sent for another set of PCR reactions in order
to determine which of the 9 possible pools of 225 contains the
knockout. After identifying the pool of interest, 25 tubes of seeds
are screened for the individual plant carrying the T-DNA
knock-out.
EXAMPLE 10
Cation Detoxification In Plant Cells
[0085] The studies described herein together with other evidence
strongly indicate that yeast and plants share pathways and signals
for the trafficking of vesicles from Golgi network to the vacuole
(Gaxiola, R., et al., Proc. Natl. Acad. Sci. USA, 96:1480-1485
(1999); Marty, F., "The Biogenesis of Vacuoles: Insights from
Microscopy. In: The Plant Vacuole, 1-42, Leigh, R. A. and Sanders,
D., Academic Press, San Diego (1997); Bassham, D.C., et al., Plant
Physiol, 117:407-415 (1998)). Without wishing to be bound by
theory, it is likely that in both systems a prevacuolar compartment
is a dynamic entity that detoxifies the cytoplasm from toxic
cations and delivers its cargo either to the vacuole, or directly
to the cell exterior. Both the Gef1 chloride channel and Nhx1
Na.sup.+/H.sup.+ exchanger have been localized to the yeast
prevacuolar compartment (Gaxiola, R. A., et al., Proc. Natl. Acad.
Sci. USA, 96:1480-1485 (1999)). The behavior of the Gef1-GFP
chimera in yeast cells in vivo have been monitored indicating that
its localization varies depending the environmental conditions.
Furthermore, it has been shown that two of the four A. thaliana CLC
chloride channel genes CLC-c and -d are capable of suppressing gef1
mutant phenotypes implying a similar localization (Gaxiola, R. A.,
et al., Proc. Natl. Acad. Sci. USA, 95:4046-4050 (1998)). In order
to understand how and where this cation detoxification takes place
in plant cells the intracellular localization of GFP chimeras of
AVP1, AtNHX1 and AtCLC-c and -d (Hong, B., et al., Plant Physiol.,
119:1165-1175 (1999)) is monitored in vivo. Confocal microscopy is
also used to address colocalization of the different transporters.
For this purpose HA-tagged versions or antibodies of the
transporters under study are required (Guiltinan, M. J., et al.,
Meth. Cell Biol., 49:143-151 (1995); Jauh, G. -Y., et al., Plant
Cell, 11:1867-1882 (1999); Mullen, R. T., et al., Plant. J.,
12:313-322 (1997)).
[0086] For the constructions of the GFP-chimeras the soluble
versions GFP with improved fluorescence in A. thaliana reported by
Davis and Viestra (Davies, S. J., Viestra, R. D., "Soluble
derivatives of green fluorescent protein (GFP) for use in
Arabidopsis thaliana, http://brindabella.mrc-lmb-
.cam.ac.uk/IndexGFP.html (1998)) are used. Two types of
GFP-chimeras are made, namely a set under the regulation of the
native promoter and another set under the regulation of the 35S
promoter. The resulting T-DNA vectors containing the GFP-chimeras
are transformed into Agrobacterium tumefaciens strain GV3101 via
electroporation, and used for subsequent vacuum infiltration of
Arabidopsis thaliana ecotype Columbia (Bechtold, N., et al., C. R.
Jeances Acad. Sci. Ser. III Sci. Vie, 316:1194-1199 (1993)). For
the hemagglutinin (HA) epitope tagging a PCR strategy designed for
yeast but modified to tag plant genes expressed in yeast vectors is
used. Futcher and coworkers designed vectors containing the URA3
yeast gene flanked by direct repeats of epitope tags (HA)
(Schneider, B. L., et al., Yeast, 11:1265-1274 (1995)). Via PCR the
tag-URA3-tag cassette is amplified such that the resulting PCR
fragment possess homology at each end to the gene of interest. In
vivo recombination in yeast is then used to direct the integration
of the PCR-chimera to the plasmid carrying the plant ORF of
interest, transformants are selected by the URA.sup.+ phenotype.
The URA3 gene is "popped out" when positive transformants are grown
in the presence of 5-fluoro-orotic acid. The vector carrying the
plant gene has a selection marker different than the URA3 gene.
EXAMPLE 1
Further Applications of the Yeast Model
[0087] Gain of function mutants of the AtNHX that enhance salt
tolerance of transgenic plants are generated using the yeast
system. This is accomplished by mutagenizing the cloned gene to
make a mutant library. This library is used to transform the salt
sensitive yeast mutant enal and clones with an enhanced salt
tolerant phenotype will be identified and retested. The other genes
that show similarity to the AtNHX1 gene reported by the Arabidopsis
Genome Initiative (AG1) are expressed in yeast. It is likely that
some of these AtNHX1 homologues are plasma membrane transporters,
so their function in yeast should be pH dependent. Thus the precise
composition and pH of the medium used for screening is crucial for
success. Identification of plasma membrane transporters helps to
engineer plants with an enhanced salt tolerance due to a reduced
sodium uptake. In addition, plant cDNA expression libraries in
yeast are used to identify other families of transporters involved
in NaCl detoxification.
[0088] To generate gain of function mutants of the AtNHX a method
for introducing random mutations developed by Stratgene (Epicurian
Coli XL1-Red competent Cells Cat#200129) is used. The method
involves the propagation of a cloned gene into a strain deficient
in the three primary DNA repair pathways. The random mutation rate
in this strain is about 5000-fold higher than that of wild-type. A
library of the mutated AtNHX gene is transformed into the enal
yeast mutant and screened for salt tolerance. Yeast transformation
is performed as described by Schiestl and coworkers (Gietz, D., et
al., Nucl Acid Res. 20:1425 (1992)). An alternative to the XL1-Red
random mutagenesis strategy is a PCR approach described by Fink and
coworkers (Madhani, H. D., et al., Cell, 91:673-684 (1997)). To
test AtNHX1 homologues the same strains and conditions used for
AtNHX1 (Gaxiola, R. A., et al., Proc. Natl. Acad. Sci. USA,
96:1480-1485 (1999)) are used initially. However, if these
screening strains and/or conditions do not work new ones are worked
out. It is likely that when dealing with plasma-membrane AtNHXl
homologues pH conditions of the assay media are crucial.
EXAMPLE 12
Hydroponic Culture of Transgenic Plants
[0089] The reduced availability of fresh water for standard
agriculture may force the use of alternative agricultural arts. It
is conceivable that with salt tolerant crops the use of hydroponics
with seawater will create a new era in crop production. As
described herein, conditions for hydroponics culture of Arabidopsis
plants have been established and their performance in increasing
concentrations of NaCl in their media have been tested. Transgenic
plants are challenged with a commercial seawater formula that
contains the complete ionic composition present in the oceans.
35SAVP1, 35SAtNHX1 single and double transgenics are grown together
with wildtype Arabidopsis thaliana plants under hydroponic
conditions for four weeks in a short day illumination cycle
(Gibeaut, D. M., et al., Plant Physiol., 317-319 (1997)). Then
every four days an equivalent to 50 mM NaCl of Tropic Marin sea
salt (www.thatpetplace.com) is added. This artificial sea water mix
includes all of the other major and trace elements present in real
sea water. Growth is monitored and physiological parameters, such
as sodium content and distribution is determined as described in
previous sections.
[0090] The effects of the overexpression of these Arabidopsis
thaliana proton transporters (AVP1 and AtNHX1) in more
agriculturally important plants such as tomato are examined. The
tomato homologues of AVP1 and AtNHX1 are isolated and the
corresponding chimeras to overexpress them are constructed (Bidone,
S., et al., Eur. J. Biochem., 253: 20-26 (1998); Burbidge, A., et
al., J. Exper. Botany, 48:2111-2112 (1997)). The genes are
introduced via Agrobacterium-mediated infection of calli. Tissue
culture methods are used to regenerate transformed plants. The
plants are assayed for salt tolerance as well as physiological
parameters, such as sodium content and distribution. Increasing the
salt-tolerance of tomato plants will likely have important economic
repercussions. A positive result indicates that the sequestration
model described herein is also applicable to an important crop.
Tomato transformation with 35S AVP1 and with 35S AtNHX1 constructs
is performed as described by McCormick (McCormick, S.,
Transformation of tomato with Agrobacterium tumefaciens. In: Plant
Tissue Culture Manual, pp. 1-9, Lindsey, K. (ed.), Kluwer Academic
Publishers, Dordrecht, The Netherlands (1991)). T0 and T1
transgenics are analyzed by polymerase chain reaction and DNA gel
blotting for the presence and copy number of AVP1 and AtNHX1
transgenes. Heterozygous and homozygous plants are identified after
segregation analysis of each transcend within T1 seeds. Homozygous
plants are assayed for salt tolerance and as well as physiological
parameters, such as sodium content and distribution. Degenerated
oligos based on conserved sequences present in AVP1 and AtNHX1
homologues are designed. These degenerated primers are used in
RT-PCR reactions with cDNAs made from poly(A)+RNA from tomato. The
resulting PCR fragments are used as probes to isolate the full
length cDNA clones from commercial libraries (i.e. Stratagene
Cat#936004). A similar strategy was described by Caboche and
coworkers (Quesada, A., et al., Plant Mol. Biol., 34:265-274
(1997)).
[0091] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *
References