Note: Descriptions are shown in the official language in which they were submitted.
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COPPER TRANSPORTER IN ETHYLENE SIGNALING PATHWAY
Reference To Related Applications
This application claims priority to US Provisional
Application 60/093,698, filed July 22, 1998.
Government Support
This work was supported in part by grants from the National
Science Foundation, grant number MCB-95-07166, the United States
Department of Energy and the National Institutes of Health, grant
number DE-FG02-93ER20104. The government may have certain rights
in this invention.
BACRGROUND OF THE INVENTION
The simple gaseous hormone ethylene (CzHA) is involved in a
variety of plant growth and developmental processes, including
germination, cell elongation, flower and leaf senescence, sex
determination and fruit ripening (Abeles et al., (1992) In
Ethylene in Plant Biology, 2°d Ed., New York, NY: Academic Press.
A number of biological stresses are known to induce ethylene
production in plants, including wounding, abscission, bacterial,
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viral or Fungal ~.nfection, and treatment with elicitors, such as
glycopeptide elicitor preparations from fungal pathogen cells.
In the case of abscission, a particular layer of cells in a zone
located between the base of the leaf stalk and the stem (the
abscission zone) responds to a complex combination of ethylene
and other endogenous plant growth regulators by a process that
is, to date, not fully understood. However, the effect of
abscission is the controlled loss of part of the plant, typically
localized; the result of which is visible as a dead leaf or
flower, or as softening or "ripening" of fruit, ultimately
leaving the plant wounded at the point of separation.
Recent studies have shown that ethylene is also required in
the determination of cell fate in the root epidermis (Tanimoto
et al., Plant J. 8:943-948 (1995)), the systematic activation of
a defense gene upon pathogen attack (Penninckx et al., Plant Cell
8:2309-2323 (1996)), the wound response in tomato (0'Donnell et
al., Science 279:1914-1917 (1996)), and the formation of
symbiotic nitrogen-fixing nodules (Penmetsa et al., Science
275:527-530 (1997)). Thus, regulated ethylene production and the
perception of ethylene by receptors in required tissues of plants
followed by signal transduction is necessary to establish,
control and maintain the complicated developmental systems found
in plants, including defensive responses to plant pathogens,
chlorosis, senescence and abscission..
The synthetic pathway of ethylene has been well
characterized (Kende, Plant Physiol., 91:1-4 (1989)). The
conversion of ACC to ethylene is catalyzed by ethylene forming
enzyme (Spanu et al., EMBO J 10:2007 (1991)). In a closed
circular ethylene synthetic pathway, S-adenosyl-1 methionine
(SAM) is produced from methionine. Then, in a rate-limiting
step, SAM is converted to 1 aminocyclopropane-1-carboxylic acid
(ACC) by an ACC synthase. In a final step, ethylene is produced
from ACC by an ACC oxidase. The pathway, therefore, utilizes
multiple ACC synthases and ACC oxidases.
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Scientists are just beginning to understand the ethylene
signal transduction pathway at the molecular level. To address
the ethylene signaling mechanisms, a molecular/genetic approach
has been applied using the ethylene-evoked triple response
phenotype of Arabidopsis thaliana seedlings. In Arabidopsis, the
"triple response" typically involves inhibition of root and stem
elongation, radial swelling of the stem and absence of normal
geotropic response (diageotropism). Etiolated morphology of a
plant can be dramatically altered by stress conditions which
induce ethylene production, so that, for example, the ethylene-
induced triple response provides a seedling with the additional
strength required to penetrate compacted soils.
Based upon the triple response, a dozen Arabidopsis mutants
have been isolated into two classes (Ecker, Science 268:667-675
(1995); Johnson & Ecker, Annu. Rev. Genetics 32:227-254 (1998):
US Pat. Nos. 5,367,065; 5,444,166; 5,602,322 and 5,650,553, each
of which is herein incorporated by reference). One class of
mutants, the ein (ethylene insensitive) mutants, show reduced or
are completely insensitive to exogenous ethylene. This group
includes etrl (Bleecker et al., Science 2Q1:1086-1089 (1988),
etr2 (Sakai et al., Proc. Natl. Acad. Sci. USA 95:5812-5817
(1998)), ein2 (Guzman & Ecker, Plant Cell 2:513-523 (1990)), ein3
(Rothenberg & Ecker, Sem. Dev. Biol. Plant Dev. Genet. 9:3-13
(1993)), ein5/ainl [Van der Straeten et al., Plant Physiol.
102:401-408 (1993)), eti (Harpham et al., Ann. Bot. 68:55-62
(1991), ein4 and ein6 (Roman et al., Genetics 139:1393-1409
(1995)). The other class of mutants, the constitutive hormone
response mutants) display constitutive ethylene response
phenotypes in the absence of exogenously applied hormones. Using
antagonists of ethylene biosynthesis and activity, this class was
further divided into hormone overproducing mutants (etol, eto2
and eto3) (Guzman and Ecker, 1990, supra; Kieber et al., Cell
72:427-441 (1993)) and into constitutive signaling mutants (ctrl;
constitutive triple response) (Kieber et al., 1993, supra.). The
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latter mutants display "ethylene" phenotypes even in the presence
of inhibitors of ethylene biosynthesis or receptor binding.
Genetic epistasis studies indicate that ETR1 and EIN4 act
upstream of CTR1, while EIN2, EIN3, EINS, EIN6 and EIN7 act
downstream of CTRL (Roman et al., 1995, supra Sakai et al.,
1998, supra; Hua et al., Plant Cell 10:1321-1332 (1998)). The
CTRI gene encodes a Raf-like serine/threonine protein kinase
(Kieber et al., 1993, supra). Raf is known as a component of a
MAP kinase cascade in mammalian cells, indicating that a MAP
kinase cascade is involved in the ethylene signaling pathway.
Given that a loss-of-function mutation in this gene confers an
ethylene constitutive response phenotype, CTRL is presumed to be
a negative regulator of the ethylene response pathway. The EIN3
gene and its related genes (EIL1, 2 and 3) have been cloned and
characterized (Chao et al., Cell 89:1133-1144 (1997)). EIN3
protein is presumed to be a new class of transcriptional
regulator as evidenced by its sequence specific DNA binding
activity in vitro (Solano et al., Genes Dev. 12:3703-3714
(1998) ) .
The mechanism of ethylene perception was first addressed by
biochemical approaches (Bleecker et al., (1997) In Biology and
Biotechnology of the Plant Hormone Ethylene, AK Kanellis, ed:
Kluweer Acad. Publ, pp. 63-70; Hua & Meyerowitz, Cell 94:261-271
(1998)). Ethylene binding assays using whole plant tissue or
organs with [14C] ethylene, revealed saturable two class binding
sites with different dissociation rates, fast dissociation (half
life, <30 minutes) and a slow dissociation (half life >6 hours)
in many plant species (reviewed by Sisler, (1990) In The Plant
Hormone Ethylene, K. Mattoo & JC Suttle, eds. (Boca Raton, FL:
CRC Press), pp. 81-100 (1990). Subsequent competitive binding
assays using ethylene antagonists supported the physiological
relevance of binding activities; however, attempts to purify the
ethylene receptors proved unsuccessful.
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Interestingly, etrl (ethylene receptor) plants display a
reduction in ethylene binding activity (Bleecker et al., 1988,
supra), indicating that ETR1 encodes a receptor for ethylene.
Mutations in the gene can cause a complete loss of the ethylene
response. Consequently, it can be concluded that the ETR1 gene
encodes a receptor for ethylene. The ETR1 gene, isolated by map-
based chromosomal walking, encodes a membrane-bound histidine
kinase (Chang et al., Science 262:539-544 (1993); Chang et al.,
Proc. Natl. Acad. Sci. USA 92):4129-4133 (1995)). The kinase is
similar to an emerging family of eukaryotic histidine kinases,
including the osmosensor of budding yeast (Wurgler-Murphy et al.,
Trends Biochem. Sci. 22:172-176 (1997)), and to sensor molecules
in the two component regulatory systems in prokaryotes.
Expression of this protein in budding yeast cells was found to
confer ethylene binding capability with similar binding
characteristics to those found in plant materials, further
confirming that the etrl gene encodes an ethylene receptor
(Schaller et al., Science 270:1809-1811 (1995)). Moreover, ETR1
protein expressed and partially purified from E. coli has been
shown to possess histidine kinase activity (Gamble et al, Proc.
Natl. Acad. Sci. USA 95.:7825-7829 (1998)).
Arabidopsis has at least four other ETRI-like genes, ERSI,
ETR2, EIN4 and ERS2 (Hua et al., 1995, supra; Hua et al., 1998,
supra; Sakai et al., 1998, supra). The ein4 and etr2 mutants
display a similar ethylene insensitive phenotype as that of etrl.
Transgenic Arabidopsis plants with an artificial mutant ERS or
ERS2 gene, which has the same mutation as etrl-I, also display
an ethylene insensitive phenotype. These results indicate that
the ETRI-related proteins are involved in the ethylene signaling
pathway. Since all identified receptor mutants are dominant, it
has been unclear until recently that receptor molecules are
positive or negative regulators of ethylene responses. Recent
studies on the loss-of-function mutations of ethylene receptor
genes have revealed that ethylene receptors negatively regulate
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the ethylene response pathway (Hua et al., 1998, supra). "Never
ripe," an ethylene insensitive mutant tomato species, has been
identified as bearing a mutation in an ERS-like gene, indicating
that the ethylene signaling pathway, at least at the perception
of this hormone, is conserved in the plant kingdom.
Copper is, of course, an essential metal for aerobic
organisms. It serves as a co-factor for enzymes such as
cytochrome c oxidase, copper-zinc superoxide dismutase, and lysyl
oxidase, and is also required for neurotransmitter synthesis_and
the maturation of connective tissues in animals (for review Uauy
et al., Am. J. Clin. Nutr. 67(suppl)e:952S-9595 (1998)).
However, paradoxically, copper is highly toxic. The intracellular
concentration of copper is tightly regulated and copper is
typically sequestered in non-reactive forms.
Nevertheless, the basic mechanisms of copper transport and
metabolism seem to be highly conserved in evolution (Askwith et
al., Trends Biochem. Sci. 23 :135-138 (1998)). In yeast, for
example, a plasma membrane-localized copper transporter imports
copper ions into the cytoplasm, where they are immediately bound
by copper trafficking proteins. Several copper distinct
trafficking proteins are known including, Atxlp, Lys7p and Coxl7,
which differ in their intracellular targets. Thus, it appears
that without a sufficient supply of copper, the receptors are
unable to assume the proper protein conformation.
By analogy with yeast, reduced copper transport by RAN1
localization into a post-Golgi compartment appears to present an
abnormal amount of copper to co-localized ethylene receptor
apoproteins (Thompson et al., J. Am. Chem. Soc. 105.:3522-3527
(1983)). This may cause altered (or relaxed) ligand specificity
allowing the recognition of both ethylene and TCO to function as
agonists. From recent studies (Rodriguez et al., Science
283:396-398 (1999)), it appears that a dimerized ethylene
receptor requires at least two molecules of copper to assume a
correct structure (and ligand specificity).
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The fact that mutations in copper trafficking genes cause
physiological disorders in many eukaryotes, supports the concept
that the proper regulation of copper metabolism is crucial. In
addition to the Menkes/Wilson disease genes (yeast
CCC2/Arabidopsis RANI), human homologues of the yeast cytosolic
copper metabolism genes have been uncovered, although not
intended to be limiting, including human Ctrl gene (a homologue
of yeast CTR1, (Zhou et al., Proc. Natl. Acad. Sci. USA 94:7481-
7486 (1997)), human Hahl gene (a homologue of yeast ATX1, (Klomp
et al., J. Biol. Chem. 272:9221-9226 (1997)), human Ccs1 gene (a
homologue to LYS7, (Culotta et al.; J. Biol. Chem. 272:23469-
23472 (1997)) and human Coxl7 gene (a homologue of yeast COX17,
(Amaravadi et al., Hum. Genetics 99:329-333 (1997)).
Because ethylene is an olefin, it has been speculated that
ethylene is perceived by a plant receptors) with transition
metals, such as a copper (Burg et al., Plant Physiol. 42:144-152
(1967). Several observations supported this idea. For example,
when silver ions (Ag(I)), which are biochemically similar to
copper ions (Cu ( I ) ) , are added to the media of plant cells in
vitro, the ethylene response is inhibited. Moreover, the
addition of either copper or silver ions increases the ethylene
binding activity of membrane extracts from yeast cells expressing
the ETR1 protein (Bleecker, 1997, supra; Bleecker et al., Science
283:996-999 (1999)). No known metal binding motifs, however, had
been reported prior to the present invention in the predicted
amino acid sequence of ETRZ or in any of its related proteins,
leaving the issue unsolved until the development of the present
invention.
The present invention, therefore, provides the novel
isolated nucleic acid, its expression product, and methods for
controlling, modulating or regulating the delivery of essential
transition metal ions, particularly copper ions, to the ethylene
receptors during the secretion pathway. By thus improving
understanding of the ethylene signal pathway, it is now possible
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to develop methods for improving the tolerance of plants to
pathogens, as well as for developing easier and more efficient
methods for identifying pathogen tolerant plants. Moreover, by
providing insight into plant hormones and mechanisms for their
control, and by modulating and regulating their functions, it is
possible to significantly improve the quality, quantity and
longevity of plant food products, such as fruits and vegetables,
flowers and flowering ornamentals, and other non-food plant
products, such as commercially valuable crops, e.g., cotton or
flax, or ornamental green plants, thereby providing more and
better products for market in both developed and~underdeveloped
countries.
SiII~IARY OF THE INVENTION
The present invention is directed to an isolated nucleic
acid encoding a plant, plant cell, tissue, flower, organ or other
plant part having copper transporter activity, and mutants,
derivatives, homologues and fragments thereof encoding a plant,
plant cell, tissue, flower, organ or other plant part having
copper transporter activity. Also included are the rant gene,
and the genomic nucleic acid sequences and cDNA comprising SEQ
ID N0:1 and 2. In addition, embodiments of the invention also
relate to a plant, plant cell, organ, flower, tissue, seed, or
progeny comprising the nucleic acid encoding a plant copper
transporter.
The invention is also directed to a purified preparation of
a polypeptide comprising a plant copper transporter in a plant,
plant cell, tissue, flower, organ or other plant part having
copper transporter activity, and analogs, derivatives and
fragments thereof having such activity. Also included in the
invention is RAN1 and the polypeptide having amino acid sequence
SEQ ID N0:3, further including an embodiment in which the copper
transporter activity is ATP-dependent.
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Further included in embodiments of the invention is a
recombinant cell comprising a plant copper transporter, a vector
comprising the isolated nucleic acid encoding a plant copper
transporter, and an antibody specific for a plant RAN1
polypeptide, analog, derivative or fragment thereof having copper
transporter activity.
Also included in the invention is an isolated nucleic acid
sequence comprising.a sequence which is complementary in an
antisense orientation to all or part of the nucleic acid encoding
a plant copper transporter sequence, and to mutants, derivatives,
homologues or fragments thereof encoding a plant, plant cell,
tissue, flower, organ or other plant part having copper
transporter activity. An embodiment further includes such
complementary nucleic acid having antisense activity at a level
sufficient to regulate, control, or modulate the copper
transporting activity of a plant, plant cell, organ, flower or
tissue. In addition, the invention also relates to a plant,
plant cell, organ, flower, tissue, seed, or progeny comprising
a nucleic acid in an antisense orientation to the nucleic acid
encoding a plant copper transporter.
The invention is further directed to a transgenic plant,
the cells, organs, flowers, tissues, seeds or progeny of which
comprise the nucleic acid encoding a plant copper transporter,
or a recombinant nucleic acid comprising the nucleic acid
encoding a plant copper transporter. Also included in the
invention is a transgenic plant, the cells, organs, flowers,
tissues, seeds or progeny of which comprise a polypeptide having
plant copper transporter activity.
In addition, the invention is directed to any of the
preceding isolated nucleic acids encoding a plant copper
~35 transporter, further comprising a plant RAN1 promoter sequence,
or a fragment thereof having promoter activity. Also included
in the invention is a vector comprising any of the preceding
isolated nucleic acids encoding a plant copper transporter.
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Moreover, an additional embodiment includes any of the preceding
isolated nucleic acids encoding a plant copper transporter,
further comprising a reporter gene operably fused thereto, or a
fragment thereof having reporter activity.
Also the invention is directed to a transgenic plant, the
cells, organs, flowers, tissues, seed, or progeny of which
comprise a transgene comprising an isolated nucleic acid
comprising a RAN1 promoter sequence.
The invention is further directed to a method for
manipulating in a plant a nucleic acid encoding a plant copper
transporter, wherein the method permits the regulation, control
or modulation of germination, cell elongation, sex determination,
flower or leaf senescence, flower maturation, fruit ripening,
insect, herbicide or pathogen resistance, abscission, or response
to stress in the plant. In one embodiment the method is directed
to the initiation or enhancement of the germination, cell
elongation, sex determination, flower or leaf senescence, flower
maturation, fruit ripening, insect, herbicide or pathogen
resistance, abscission, or response to stress of the plant, in
another embodiment the method is directed to the inhibition or
prevention of such responses in the plant, plant cell, organ,
flower or tissue.
In addition, the invention is directed to a method of
identifying a compound capable of affecting the transport of
copper in the ethylene signaling system in a plant comprising:
providing a cell comprising an isolated nucleic acid encoding a
plant RAN1 sequence, having a reporter sequence operably linked
thereto; adding to the cell a compound being tested; and
measuring the level of reporter gene activity in the cell,
wherein a higher or lower level of reporter gene activity in the
cell compared with the level of reporter gene activity in a
second cell to which the compound being tested was not added, is
an indicator that the compound being tested is capable of
affecting the expression of a plant ranl gene.
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Moreover, the invention is directed, in one embodiment, to
a method for generating a modified plant with enhanced copper
transporting activity as compared to that of comparable wild type
plant comprising introducing into the cells of the modified plant
an isolated nucleic acid encoding RAN1, wherein said ranl nucleic
acid is capable of transporting copper within the cells of the
modified plant. Another embodiment is directed to a method for
generating a plant with diminished or inhibited copper
transporting activity as compared to that of a comparable wild
type plant comprising binding or inhibiting the copper
transporting molecules within the cells of the modified plant by
introducing into said cells an isolated nucleic acid encoding a
complementary nucleic acid to all or a portion of ranl, wherein
said ranl nucleic acid would otherwise be capable of transporting
copper within the cells of the modified plant. An additional
embodiment provides a method for generating a plant with
diminished or inhibited copper transporting activity comprising
binding or inhibiting the copper transporting molecules within
the cells of the modified plant by introducing into said cells
an antibody to all or a portion of RAN1, wherein said RAN1
polypeptide would otherwise be capable of transporting copper
within the cells of the modified plant.
Further, the invention is directed to a method for
manipulating the expression of RAN1 in a plant cell comprising
operably fusing the nucleic acid ranl or an operable portion
thereof to a plant promoter sequence in the plant cell to form
a chimeric DNA, and generating a transgenic plant, the cells of
which comprise said chimeric DNA, where upon controlled
activation of the plant promoter, manipulates expression of RAN1.
The invention is also directed to the nucleic acid
encoding and the polypeptide expression products of mutant
alleles ranl-1 and ranl-2.
Additional objects, advantages and novel features of the
invention will be set forth in part in the description, examples
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and figures which follow, and in part will become apparent to
those skilled in the art on examination of the following, or may
be learned by practice of the invention.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 is a model representing the function of RAN1 in the
ethylene signal transduction pathway. Filled and unfilled shapes
indicate the active and inactive states of signaling pathway
components, respectively.
Figs. 2A and 2B illustrate the phenotype of the rant
mutants. Fig. 2A shows the seedling phenotype of wild type
plants (Col), ranl-1, ranl-2, and ethylene over-producing mutant
(etol-5) and constitutive triple response mutant (ctrl-1) in air,
transcyclooctene (TCO) or ethylene (CZH9). Fig. 2B shows the
rosette plant phenotype, in air and in TCO.
Figs . 3A and 3B illustrate that ethylene inducible genes
are up-regulated in the ranl mutants by TCO treatments. In Fig.
3A, northern blots are shown of total RNA prepared from seedlings
treated with hydrocarbon-free air, ethylene or TCO. Arabidopsis
a-tublin 3 (TUA3) was used as a control. The same blot was used
in three hybridizations. In Fig. 3B, an analysis is shown of the
expression of the basic chitinase gene, CHIB, in TCO-treated
adult plants. Northern blot analysis was done using total RNA
prepared from rosette leaves exposed to hydrocarbon-free air or
TCO for three (3) days.
Fig. 4 illustrates the fine mapping of the RANT gene to
160 kb region at the bottom of chromosome 5 by the
identification of three (3) each recombinant chromosomes using
the simple sequence length polymorphism (SSLP) markers CIC2AT2L
and CIC4ET2R. The assembled BAC contig covering this region is
shown. Additionally, new markers developed from the sequence of
T19K24 permitted localization of the RAN1 gene on a single BAC
(bacterial artificial chromosome). The predicted gene
organization of the BAC insert is drawn according to .the
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annotation of T19K24 (GenBank AC002342). Gray or black boxes
represent predicted genes. The intron and exon organization of
the RAN1 gene is shown at the bottom. Arrowheads indicate the
locations of the ranl-1 and ranl-2 mutations.
Fig. 5 illustrates the genomic nucleotide sequence of the
RANI gene, SEQ ID N0:1; GenBank accession #AF091112. Beneath the
nucleotide sequence is the predicted amino acid sequence, SEQ ID
N0:3. The upper case and lower case characters indicate exon and
intron sequences, respectively. The exon-intron boundaries were
determined by comparison with the sequence of cDNA, SEQ ID N0:2,
GenBank acession # AF082565, which extends from nucleotide -275
through nucleotide 4201. The numbers for the nucleotides are
shown on the left, with the A from the first ATG codon assigned
the number 1. The amino acid numbers are also indicated.
Asterisks indicate in-frame stop codons.
Fig. 6A shows a Fet3p oxidase assay in which oxidase
activity of the membrane fraction from various yeast cells shown
in panel A was measured using an in-gel assay system. The lower
panel shows the result of a western blot of membrane fraction
samples used in the oxidase assay using anti-HA-tag antibody.
Fig. 6B shows a high affinity iron uptake assay in which the
iron uptake activity of wild-type or ccc2-disrupted yeast cells
expressing various proteins were measured. This included yeast
wild-type CCC2, RAN1:HA-tag, ranl-l:HAtag, or ranl-2:HAtag.
Averages from three (3) independent experiments are shown. Error
bars indicate standard deviations.
Fig. 7 illustrates that copper partially suppresses the
rant phenotype. The seeds of various strains were germinated on
MS medium absent added copper ions (- CuS04) or on the same
medium containing 12.5 uM CuS04 (+ CuS04). Both were exposed to
hydrocarbon-free air or TCO. After three (3) days of incubation,
the seedlings were photographed. The applied concentration of
CuS04 had no effect on the triple response of etol-5 or ctrl-1
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mutants grown in hydrocarbon-free air or on the growth-promoting
activity of TCO treatment of etol-5 seedlings.
Figs. 8A - 8C illustrate the constitutive ethylene response
phenotypes in 35S::RAN1 transgenic plants. Fig. 8A shows the
phenotype of 35S::RAN1 transgenic plants, specifically three-week
l0 old transgenic plants. Approximately one-half of the transgenic
plants displayed a ctrl-like phenotype (class 1), while the other
half displayed normal morphology (class 2). The photographs were
taken at the same magnification. Fig. 8B shows the expression
of RAN1 (upper panel) and the ethylene-inducible basic-chitinase
(CHIB) gene (lower panel) in transgenic plants. Northern blots
were performed using 20 ug of total RNA per lane from adult
plants. In the class 1 transgenic plants, RNA was prepared from
five (5) independent Tl-generation plants and loaded in one lane.
Fig. 8C shows the ethylene response phenotypes in the transgenic
seedlings. Representative dark-grown T2-generation seedlings are
shown from ranl-2 and L1 (35S::RAN1) transgenic plants treated
with TCO for three days, Also shown are representative dark-
grown T2-generation seedlings from 35S::RAN1-containing plants
(L6 and L3) and seedlings from the ctrl-1 mutant treated with
hydrocarbon-free air for three days.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based upon the identification, pursuant to
a novel screen for antagonist responsive mutants, of an early
acting, negative regulator in the ethylene gas signaling pathway
in Arabidopsis. As a result of the present invention, new
insights into the mechanisms involved in the ethylene signaling
pathway are evident. To better understand the mechanisms
underlying ethylene perception, the present inventors have
isolated mutants in which ethylene recognition is changed. In
doing so, mutants have been identified and characterized in the
present invention that show an altered ligand specificity of
their ethylene receptors.
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An ethylene antagonist, traps-cyclooctene (TCO), activates
the ethylene signaling pathway in the class of mutants of this
invention, as opposed to inhibiting or blocking the pathway. The
mutants are encoded by alleles of ranl (responsive to antagonist
1). Map based cloning of the RAN1 gene by the inventors revealed
ranl to be a homologue of human Menkes-Wilson disease genes and
of the yeast CCC2 gene. The RAN1 protein has been found to
advantageously provide copper transporting activity to plant
cells. Coincidentally, recent studies of the human Menkes
protein, Wilson protein and yeast Ccc2p suggest that those
proteins may also have a copper transporter function and deliver
copper ions into the secretion pathway.
In accordance with the present invention, genetic,
molecular and biochemical studies of RAN1 provide the first in
vivo evidence of a requirement of copper ions for ethylene
perception/signal transmission in plants. Moreover, functional
studies of RAN1 demonstrate that the mechanisms controlling
copper trafficking in yeast and plant cells are conserved.
Discovery of the requirement of RAN1/copper for the assembly of
functional hormone receptors facilitate an understanding of the
mechanism of ethylene perception in plants and offers a useful
paradigm for copper-dependent signaling processes in other
organisms.
To identify mutations in novel components of the ethylene
gas signal transduction pathway, a screen was initiated for
Arabidopsis thaliana mutants that exhibited an ethylene-like
triple response phenotype in response to a potent hormone
antagonist. The "triple response" in Arabidopsis consists of
three distinct morphological changes in dark-grown seedlings upon
exposure to ethylene: 1) inhibition of hypocotyl and root
elongation, 2) radial swelling of the stem and 3) exaggeration
of the apical hook. A class of constitutive mutants, ctr,
display a constitutive triple response in the presence of
ethylene biosynthetic inhibitors, and is most likely affected at,
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or downstream of the receptor. Based on the results of genetic
experiments, over-expression of the normal or truncated versions
of the regulatory gene ranl in transgenic plants was identified
by the inventors and characterized as an early acting gene in the
ethylene response pathway, displaying an ethylene triple response
phenotype in response to potent receptor antagonists.
It should be appreciated that the present invention is not
limited by the proposed models and mechanisms described herein.
Thus, it should be understood that models such as the one shown
in Fig. 1, for example, present a working model showing the role
of RAN1 to deliver copper ions to create functional ethylene
receptors, and the like, in the ethylene signal transduction
pathway, all of which facilitates understanding of the invention.
RAN1 is shown as localized on the membrane of post-Golgi
compartments. RAN1 receives copper ions from copper chaperons
such as Atx1-like proteins and transports them into a post-Golgi
compartment, delivering the metal to membrane-targeted ethylene
receptors. After the incorporation of copper ion, the receptors
are localized on the plasma membrane, where they functionally
coordinate ethylene.
Ethylene is an olefin, therefore its receptors are presumed
to require coordination of a transition metal for hormone binding
activity. In a preferred embodiment of the present invention,
the transition metal is a silver ion (Ag (I)). In a particularly
preferred embodiment the transition metal is a copper ion (Cu
(I)). Either these or other transition metals are incorporated
into the ethylene receptors. Once there, they may inhibit
ethylene binding or proper folding of a functional receptor,
resulting in constitutively active receptor proteins.
Reduction of copper transport causes the Ran- phenotype.
Thus, it appears that without a sufficient supply of copper, the
receptors are unable to assume the proper protein conformation.
In wild type plants, ethylene binding to its receptor
inactivates the activity of ethylene receptors (presumably
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causing a reduction in the histidine kinase activity), and
consequently causing induction of the ethylene response through
activation (de-repression) of the signaling pathway. In the
absence of copper delivery by RANT in co-suppressed transgenic
plants (class 1), the ethylene receptors are nonfunctional
(absent of kinase activity). Consequently, even in the absence
of ethylene, the hormone response pathway is constitutively
activated.
Loss-of-function ethylene receptor mutants have been shown
to function as negative regulators of the signaling pathway and
show significant functional overlap. Moreover, binding of
ethylene to the receptors) presumably inhibits biochemical
activity. More severe alterations in the expression of RANG
causes partial or complete activation of ethylene responsive
genes and phenotypes. Furthermore, the phenotypes observed in
RAN1 co-suppressed plants were indistinguishable from those
described for the quadruple ethylene receptor knockout mutants.
The resulting plants were severely stunted, and fully infertile.
(Hua & Meyerowitz, 1998, supra), confirming that copper is
required by more than one ethylene receptor for functionality.
RAN1 activity appears to be required for the functionality
of at least four or more of the ethylene receptors. Thus, loss
of expression of this single critical regulator of receptor
function results in de-repression of the entire ethylene response
pathway. Copper is required for assembly of the receptor
complex, and in its absence, these proteins appear to be turned
over more rapidly (i.e., none of the ethylene receptors may be
functional).
By "plant" as used herein, is meant any plant and any part
of such plant, wild type, treated, genetically manipulated or
recombinant, including transgenic plants. The term broadly
refers to any and all parts of the plant, including plant cell,
tissue, flower, leaf, stem, root, organ, and the like, and also
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including seeds, progeny and the like, whether such part is
specifically named or not.
By "copper transporter activity" or "copper transporting
activity" as used herein, is meant a protein or polypeptide which
transports copper or other transition metals to the post-Golgi
region, after which the metal is delivered to membrane-targeted
ethylene receptors in the ethylene signaling pathway. Although
reference is made by~example to "copper" in the delivery system,
the term is meant to exemplify any transition metal involved in
the secretory pathway.
As used herein "biologically active" refers to copper
transporting activity, meaning a polypeptide or fragment thereof
which is capable of transporting a copper ion in the ethylene
signal system as shown in the model of the present invention.
At the molecular, cellular and whole plant level, and in
seedling and adult plants, air-grown ranl mutants appear
identical to ethylene-treated wild-type plants. The dominant
nature of ranl suggests that the ethylene-response pathway is
normally under positive regulation and loss of function of the
RAN1 repressing activity by treatment with TCO results in
activation of the constitutive triple response phenotype.
The gene corresponding to RAN1 has been cloned as set forth
below and the sequence of cDNA clone is described.
Physiological, biochemical and genetic evidence indicate that the
ranl gene product is required for transduction of the ethylene
signal in both etiolated seedling and adult plants. The putative
RAN1 polypeptide acts as a positive regulator in the ethylene
signal transduction chain, while exposure to the ethylene
receptor antagonist trans-cyclooctene (TCO) prevents this
morphological transformation. Instead of acting as a competitive
inhibitor of ethylene binding in ranl seedlings, TCO treatment
causes activation of the triple response, mimicking the effects
of ethylene.
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A reduced delivery of copper ions to the ethylene receptors
appears to produce a state of suboptimal copper . apoprotein
stoichiometry. Such an altered protein conformation results in
reduced ligand specificity, thereby allowing TCO, as well as
ethylene, to act as agonists. Analogous to multi-receptor
ZO knockout mutants, severe reduction of RAN1 function results in
copper-depletion of all ethylene receptors, causing de-repression
of the signaling pathway and activation of all known ethylene
response phenotypes.
Several ranl alleles have been identified, as exemplified
by ranl-1 and ranl-2. Such mutants are recessive and modulate
ethylene perception. Sequence analysis of the exemplified ranl
mutant alleles revealed that both mutations cause amino acid
substitutions in residues found within conserved protein motifs
(metal-binding and ATPase domains), suggesting reduced function.
Expression of the mutant rant proteins in the yeast ccc2 mutant
was insufficient to restore full Fet3p activity, supporting the
understanding that ranl alleles are partial loss-of-function
mutations. Nevertheless, in view of the descriptions provided,
it is understood that other alleles and variations would be
available to one of ordinary skill in the art. Therefore,
additional mutants are also enabled by the present invention that
have insertions, deletions, alterations or substitutions within
the same conserved protein motif, so long as copper transporting
activity is expressed or regulated.
Additional evidence for the in vivo requirement of copper
for ethylene perception/signaling is provided by studies of the
rant mutants (see the Examples). Based on several lines of
evidence, the ranl mutants are hypomorphs, effecting a reduction
in copper-transporting activity. The ranl-1 and ranl-2 alleles
contain a miss-sense mutation in the phosphate and metal binding
motifs, respectively. Thus, the conclusion that a defect in a
copper transporter causes altered ligand specificity of ethylene
receptors is supported by the findings that the defect in the
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rant-1 and ranl-2 mutants allows response to trans- but not cis-
cyclooctene (CCO) or 1-methylcyclopropene (MCP), potent
competitive inhibitors of ethylene-receptor binding.
In sum, the invention should be construed to include
nucleic acid comprising ranl, or any mutant, derivative,
homologue or fragment thereof, which encodes a copper transporter
of the ethylene signaling pathway. In accordance with the
present invention, nucleic acid sequences include, but are not
limited to DNA, including and not limited to cDNA and genomic
DNA: RNA, including and not limited to mRNA and tRNA, and may
include chiral or mixed molecules.
Preferred nucleic acid sequences include, for example,
those set forth in SEQUENCE ID NOS: 1 and 2, as well as
modifications in those nucleic acid sequences, including
alterations, insertions, deletions, mutations, homologues and
fragments thereof encoding a regulatory protein in the ethylene
response pathway affecting copper transporter activity in the
ethylene response pathway.
A "fragment" of a nucleic acid is included within the
present invention if it encodes substantially the same expression
product as the isolated nucleic acid, or if it encodes a peptide
having copper transporter capability.
The invention should also be construed to include peptides,
polypeptides or proteins comprising RANT, or any mutant,
derivative, varient, analogs, homologue or fragment thereof,
having copper transporter activity in the ethylene signaling
pathway. The terms "protein," "peptide," "polypeptide," and
"protein sequences" are used interchangeably within the scope of
the present invention, and include, but are not limited to the
sequence set forth in SEQUENCE ID N0: 3, the amino acid~sequences
corresponding to nucleic acid SEQUENCE ID NOS: 1 and 2, as well
as those sequences representing mutations, derivatives, analogs
or homologues or fragments thereof having copper transporter
activity in the ethylene response pathway.
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The invention also provides for analogs of proteins,
peptides or polypeptides encoded by the gene of interest,
preferably rant. "Analogs" can differ from naturally occurring
proteins or peptides by conservative amino acid sequence
differences or by modifications which do not affect sequence, or
by both. "Homologues" are chromosomal DNA carrying the same
genetic loci; when carried on a diploid cell there is a copy of
the homologue from each parent.
For example, conservative amino acid changes may be made,
which although they alter the primary sequence of the peptide,
do not normally alter its function. Conservative amino acid
substitutions of this type are known in the art, e.g., changes
within the following groups: glycine and alanine; valine,
isoleucine and leucine; aspartic acid and glutamic acid;
asparagine and glutamine; serine and threonine; lysine and
arginine; or phenylalanine and tyrosine.
Modifications (which do not normally affect the primary
sequence) include in vivo or in vitro chemical derivatization of
the peptide, e.g., acetylation or carbonation. Also included are
modification of glycosylation, e.g., modifications made to the
glycosylation pattern of a polypeptide during its synthesis and
processing, or further processing steps. Also included are
sequences in which amino acid residues are phosphorylated, e.g.,
phosphotyrosine, phosphoserine or phosphothreonine.
Also included in the invention are polypeptides which have
been modified using ordinary molecular biology techniques to
improve their resistance to proteolytic degradation or to
optimize solubility or to render them more effective as a
therapeutic agent. Analogs of such peptides include those
containing residues other than the naturally occurring L-amino
acids, e.g., D-amino acids or non-naturally occurring synthetic
molecules. However, the polypeptides of the present invention
are not intended to be limited to products of any specific
exemplary process defined herein.
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"Derivative" is intended to include both functional and
chemical derivatives, including fragments, segments, variants or
analogs of a molecule. A molecule is a "chemical derivative" of
another, if it contains additional chemical moieties not normally
a part of the molecule. Such moieties may improve the molecule's
solubility, absorption, biological half life, and the like, or
they may decrease toxicity of the molecule, eliminate or
attenuate any undesirable side effect of the molecule, and the
like. Moieties capable of mediating such effects are disclosed
in Remington's Pharmaceutical Sciences (1980). Procedures for
coupling such moieties to a molecule are well known in the art.
Included within the meaning of the term "derivative" as used in
the present invention are "alterations," "insertions," and
"deletions" of nucleotides or peptides, polypeptides or the like.
A "variant" or "allelic or species variant" of a protein
refers to a molecule substantially similar in structure and
biological activity to the protein. Thus, if two molecules
possess a common activity and may substitute for each other, it
is intended that they are "variants," even if the composition or
secondary, tertiary, or quaternary structure of one of the
molecules is not identical to that found in the other, or if the
amino acid or nucleotide sequence is not identical.
A "fragment" of a polypeptide is included within the
present invention if it retains substantially the same activity
as the purified peptide, or if it has copper transporting
activity. Such fragment of a peptide is also meant to define a
fragment of an antibody.
In accordance with the invention, the RAN1 and ranl nucleic
acid sequences employed in the invention may be exogenous
sequences. Exogenous or heterologous, as used herein, denotes
a nucleic acid sequence which is not obtained from and would not
normally form a part of the genetic makeup of the plant, cell,
organ, flower or tissue to be transformed, in its untransformed
state. Plants comprising exogenous nucleic acid sequences of
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RAN1 or ranl mutations are encoded by, but not limited to, the
nucleic acid sequences of SEQUENCE ID NOS: 1 and 2, including
alterations, insertions, deletions, mutations, homologues and
fragments thereof.
Transformed plant cells, tissues and the like, comprising
nucleic acid sequences of RAN1 or rant mutations, such as, but
not limited to, the nucleic acid sequences of SEQUENCE ID NOS:
1 and 2, are within the scope of the invention. Transformed
cells of the invention may be prepared by employing standard
transformation techniques and procedures as set forth in Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
By the term "nucleic acid encoding" the plant cell and the
like having copper transporter activity, as used herein is meant
a gene encoding a polypeptide capable of transporting copper as
described above. The term is meant to encompass DNA, RNA, and
the like.
As described in the following Examples, ranl genes encode
proteins having specific domains located therein, for example,
terminal extensions, transmembrane spans, TM1 and TM2, nucleotide
binding folds, a putative regulatory domain, and the C-terminus.
A mutant, derivative, homologue or fragment of the subject gene
is, therefore also one in which selected domains in the related
protein share significant homology (at least about 40$ homology)
with the same domains in the preferred embodiment of the present
invention. It will be appreciated that the definition of such
a nucleic acid encompasses those genes having at least about 400
homology, in any of the described domains contained therein.
In addition, when the term "homology" is used herein to
refer to the domains of these proteins, it should be construed
to be applied to homology at both the nucleic acid and the amino
acid levels. Significant homology between similar domains in
such nucleic acids or their protein products is considered to be
at least about 40%, preferably, the homology between domains is
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at least about 50$, more preferably, at least about 60g, even
more preferably, at least about 70~, even more preferably, at
least about 80~, yet more preferably, at least about 90~ and most
preferably, the homology between similar domains is about 99~,
or the protein products thereof.
According to the present invention, preferably, the
isolated nucleic acid encoding the biologically active copper
transporter polypeptide or fragment thereof of the ethylene
signal system is full length or of sufficient length to encode
a regulated or active copper transporter. In one embodiment the
nucleic acid is at least about 200 nucleotides in length. More
preferably, it is at least 400 nucleotides, even more preferably,
at least 600 nucleotides, yet more preferably, at least 800
nucleotides, and even more preferably, at least 1000 nucleotides
in length. In another embodiment, preferably, the purified
preparation of the isolated polypeptide having copper transporter
polypeptide activity in the ethylene signal system is at least
about 60 amino acids in length. More preferably, it is at least
120 amino acids, even more preferably, at least 300 amino acids,
yet more preferably, at least 500 amino acids, and even more
preferably, at least 700 amino acids in length. In an additional
embodiment the polypeptide encodes the full length RAN1 protein
or a regulated version thereof.
The invention further includes a vector comprising a gene
encoding RAN1. DNA molecules composed of a protein gene or a
portion thereof, can be operably linked into an expression vector
and introduced into a host cell to enable the expression of these
proteins by that cell. Alternatively, a protein may be cloned
in viral hosts by introducing the "hybrid" gene operably linked
to a promoter into the viral genome. The protein may then be
expressed by replicating such a recombinant virus in a
susceptible host. A DNA sequence encoding a protein molecule may
be recombined with vector DNA in accordance with conventional
techniques. When expressing the protein molecule in a virus, the
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hybrid gene may be introduced into the viral genome by techniques
well known in the art. Thus, the present invention encompasses
the expression of the desired proteins in either prokaryotic or
eukaryotic cells, or viruses which replicate in prokaryotic or
eukaryotic cells.
Preferably, the proteins of the present invention are
cloned and expressed in a virus. Viral hosts for expression of
the proteins of the present invention include viral particles
which replicate in prokaryotic host or viral particles which
infect and replicate in eukaryotic hosts. Procedures for
generating a vector for delivering the isolated nucleic acid or
a fragment thereof, are well known, and are described for example
in Sambrook et al., supra. Suitable vectors include, but are not
limited to, disarmed Agrobacterium tumor inducing (Ti) plasmids
(e.g., pBINl9) containing a target gene under the control of a
vector, such as the cauliflower mosaic (CaMV) 35S promoter
(Lagrimini et al, Plant Cell 2:7-18 (1990)) or its endogenous
promoter (Bevan, Nucl. Acids Res. 12:8711-8721(1984)),
adenovirus, bovine papilloma virus, simian virus, tobacco mosaic
virus and the like.
Once the vector or DNA sequence containing the constructs
has been prepared for expression, the DNA constructs may be
introduced or transformed into an appropriate host. Various
techniques may be employed, such as protoplast fusion, calcium
phosphate precipitation, electroporation, or other conventional
techniques. As is well known, viral sequences containing the
"hybrid" protein gene may be directly transformed into a
susceptible host or first packaged into a viral particle and then
introduced into a susceptible host by infection. After the cells
have been transformed with the recombinant DNA (or RNA) molecule,
or the virus or its genetic sequence is introduced into a
susceptible host, the cells are grown in media and screened for
appropriate activities. Expression of the sequence results in
the production of the protein of the present invention.
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Procedures for generating a plant cell, tissue, flower,
organ or a fragment thereof, are well known in the art, and are
described, for example, in Sambrook et al., supra. Suitable
cells include, but are not limited to, cells from yeast,
bacteria, mammal, baculovirus-infected insect, and plants, with
applications either in vivo, or in tissue culture. Also included
are plant cells transformed with the gene of interest for the
purpose of producing cells and regenerating plants having
modulated copper transporting capability.
Suitable vector and plant combinations will be readily
apparent to those skilled in the art and can be found, for
example, in Maliga et al., 1994, Methods in Plant Molecular
Biology: A Laboratory Manual, Cold Spring Harbor, New York).
Transformation of plants may be accomplished using
Agrobacterium-mediated leaf disc transformation methods described
by Horsch et al., 1988, Leaf Disc Transformation: Plant Molecular
Biology Manual A5:1). Numerous procedures are known in the art
to assess whether a transgenic plant comprises the desired DNA,
and need not be reiterated.
The expression of the desired protein in eukaryotic hosts
requires the use of eukaryotic regulatory regions. Such regions
will, in general, include a promoter region sufficient to direct
the initiation of RNA synthesis. Preferred eukaryotic promoters
include, but are not limited to, the SV40 early promoter (Benoist
et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene
promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-
6975 (1982)) and the exemplified pYES3 PGK1 promoter. As is
widely known, translation of eukaryotic mRNA is initiated at the
codon which encodes the first methionine. For this reason, it
is preferable to ensure that the linkage between a eukaryotic
promoter and a DNA sequence which encodes the desired protein
does not contain any intervening codons which are capable of
encoding a methionine (i.e., AUG).
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The desired protein encoding sequence and an operably
linked promoter may be introduced into a recipient prokaryotic
or eukaryotic cell either as a non-replicating DNA (or RNA)
molecule, which may either be a linear molecule or, more
preferably, a closed covalent circular molecule. Since such
molecules are incapable of autonomous replication, the expression
of the desired protein may occur through the transient expression
of the introduced sequence. Alternatively, permanent expression
may occur through the integration of the introduced sequence into
the host chromosome. For expression of the desired protein in
a virus, the hybrid gene operably linked to a promoter is
typically integrated into the viral genome, be it RNA or DNA.
Cloning into viruses is well known and thus, one of skill in the
art will know numerous techniques to accomplish such cloning.
Cells which have stably integrated the introduced DNA into
their chromosomes can be selected by also introducing one or more
reporter genes or markers which allow for selection of host cells
which contain the expression vector. The reporter gene or marker
may complement an auxotrophy in the host (such as leu2, or ura3,
which are common yeast auxotrophic markers), biocide resistance,
e.g., antibiotics, or resistance to heavy metals, such as copper,
or the like. The selectable marker gene can either be directly
linked to the DNA gene sequences to be expressed, or introduced
into the same cell by co-transfection.
Additional elements may also be needed for optimal
synthesis of mRNA. These elements may include splice signals,
as well as transcription promoters, enhancers, and termination
signals. The cDNA expression vectors incorporating such elements
include those described by Okayama, H., Mol. Cell. Biol. 3:280
(1983), and others.
In another embodiment, the introduced sequence will be
incorporated into a plasmid or viral vector capable of autonomous
replication in the recipient host cell. Any of a wide variety
of vectors may be employed for this purpose. Factors of
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importance in selecting a particular plasmid or viral vector
include: the ease with which recipient cells that contain the
vector may be recognized and selected from those recipient cells
which do not contain the vector; the number of copies of the
vector which are desired in a particular host; and whether it is
desirable to be able to "shuttle" the vector between host cells
of different species.
The invention further defines methods for manipulating the
nucleic acid in a plant to permit the regulation, control or
modulation of germination, abscission, cell elongation, sex
determination, flower or leaf senescence, flower maturation,
fruit ripening, insect, herbicide or pathogen resistance, or
response to stress in said plant. In a preferred embodiment the
method initiates or enhances the above responses; whereas in
another preferred embodiment the method inhibits or prevents the
above responses.
Thus, methods of the present invention define embodiments
in which the copper transporting activity is prevented or
inhibited. By "prevention" is meant the cessation of copper
transport activity in the ethylene signal pathway. By
"inhibition" is meant a statistically significant reduction in
the amount of copper transport activity or RAN1 protein detected
as compared with plants, plant cells, organs, flowers or tissues
grown without the inhibitor or disclosed method of inhibition.
Preferably, the inhibitor reduces copper transport by at least
20 ~, more preferably by at least 50~, even more preferably by
80~ or greater, and also preferably, in a dose-dependent manner.
Once inhibitors satisfying these requirements are identified,
the utilization of assay procedures to identify the manner in
which the copper transport is inhibited are particularly useful.
Similarly, methods of the present invention are defined in
which the copper transporting activity is initiated, stimulated
or enhanced if there is a statistically significant increase in
the amount of copper transport activity or RAN1 protein detected
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as compared with plants, plant cells, organs, flowers or tissues
grown without the enhancer or disclosed method of enhancement.
Preferably, the enhancer increases copper transport by at least
20$, more preferably by at least 50~, even more preferably by 80~
or greater, and also preferably, in a dose-dependent manner.
Once enhancers satisfying these requirements are identified, the
utilization of assay procedures to identify the manner in which
the copper transport is enhanced are particularly useful.
The invention further features an isolated preparation of
a nucleic acid which is antisense in orientation to a portion or
all of rant or of a plant copper transporter gene. The antisense
nucleic acid should be of sufficient length as to inhibit
expression of the target gene of interest. The actual length of
the nucleic acid may vary, depending on the target gene, and the
region targeted within the gene. Typically, such a preparation
will be at least about 15 contiguous nucleotides, more typically
at least 50 or even more than 50 contiguous nucleotides in
length.
As used herein, a sequence of nucleic acid is considered to
be antisense when the sequence being expressed is complementary
to, and essentially identical to the non-coding DNA strand of the
ranl gene, but which does not encode RAN1. "Complementary"
refers to the subunit complementarity between two nucleic acids,
e.g., two DNA molecules. When a nucleotide position in both
molecules is occupied by nucleotides normally capable of base
pairing with each other, then the nucleic acids are said to be
complementary to each other. Thus two nucleic acids are
complementary when a substantial number (at least 50~) of the
corresponding positions in each of the molecules are occupied by
nucleotides which normally base-pair with each other (e.g., A:T
and G:C nucleotide pairs.).
In yet another aspect of the invention, antibodies are
provided which are directed against the copper transporter
polypeptide of the ethylene signaling system, preferably RAN1,
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which antibody is specific for the whole molecule, its N- or C-
terminal, or internal portions. Methods of generating such
antibodies are well known in the art.
In the embodiment directed to the antibody specific for a
plant RAN1 polypeptide, including functional equivalents of the
l0 antibody, the term "functional equivalent" refers to any molecule
capable of specifically binding to the same antigenic determinant
as the antibody, thereby neutralizing the molecule, e.g.,
antibody-like molecules, such as single chain antigen binding
molecules.
The invention further includes a transgenic plant
comprising an isolated DNA encoded plant copper transporter or
active fragment thereof, capable of transporting copper in the
ethylene signaling system. For instance provided in at least one
example if the current invention, a transgenic Arabidopsis plant
comprising a yeast cccLl transgene rescued by the addition of the
ranl, which when expressed confers upon the plant the ability
recognize the presence of ethylene, an ability that had been
deleted from the original yeast gene.
By "transgenic plant" as used herein, is meant a plant,
plant cell, tissue, flower, organ, including seeds, progeny and
the like, or any part of a plant, which comprise a gene inserted
therein, which gene has been manipulated to be inserted into the
plant cell by recombinant DNA technology. The manipulated gene
is designated a "transgene." The "nontransgenic," but
substantially homozygous "wild type plant" as used herein, means
a nontransgenic plant from which the transgenic plant was
generated. The transgenic transcription product may also be
oriented in an antisense direction as describe above.
The generation of transgenic plants comprising sense or
antisense DNA encoding the copper transporter of the ethylene
signaling pathway, preferably RAN1, may be accomplished by
transforming the plant with a plasmid, liposome, or other vector
encoding the desired DNA sequence. Such vectors would, as
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described above, include, but are not limited to the disarmed
Agrobacterium tumor-inducing (Ti) plasmids containing a sense or
antisense strand placed under the control of a strong
constitutive promoter, such as 35CaMV 35S or under an inducible
promoter. Methods of generating such constructs, plant
transformation and plant regeneration methods are well known in
the art once the sequence of the gene of interest is known, for
example as described in Ausubel et al., 1993, Current Protocols
in Molecular Biology, Greene & Wiley, New York).
In accordance with the present invention, plants included
within its scope include both higher and lower plants of the
Plant Kingdom. Mature plants, including rosette stage plants,
and seedlings are included in the scope of the invention. A
mature plant, therefore, includes a plant at any stage in
development beyond the seedling. A seedling is a very young,
immature plant in the early stages of development.
Transgenic plants are also included within the scope of the
present invention, having a phenotype characterized by the RAN1
gene or ranl mutations.
Preferred plants of the present invention, which are
affected by the copper transporter in the ethylene signal system
include, but are not limited to, high yield crop species for
which cultivation practices have already been perfected
(including monocots and dicots, e.g., alfalfa, cashew, cotton,
peanut, fava bean, french bean, mung bean, pea, walnut, maize,
petunia, potato, sugar beet, tobacco, oats, wheat, barley and the
like), or engineered endemic species. Particularly preferred
plants are those from: the Family Umbelliferae, particularly of
the genera Daucus (particularly the species carota, carrot) and
Apium (particularly the species graveolens dulce, celery) and the
like; the Family Solanacea, particularly of the genus
.Lycopersicon, particularly the species esculentum (tomato) and
the genus Solanum, particularly the species tuberosum (potato)
and melongena (eggplant), and the like, and the genus Capsicum,
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particularly the species annum (pepper) and the like; and the
Family Leguminosae, particularly the genus Glycine, particularly
the species max (soybean) and the like; and the Family
Cruciferae, particularly of the genus Brassica, particularly the
species campestris (turnip), oleracea cv Tastie (cabbage),
oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor
(broccoli) and the like; the Family Compositae, particularly the
genus Lactuca, and the species satira (lettuce), and the genus
Arabidopsis, particularly the species thaliana (Thale cress) and
the like. Of these Families, the more preferred are the leafy
vegetables, for example, the Family Cruciferae, especially the
genus Arabidopsis, most especially the species thaliana.
Preferred plants particularly include flowering plants,
such as roses, carnations, chrysanthemums and the like, in which
longevity of the flower on the stem (delayed abscission) is of
particular relevance, and especially include ornamental flowering
plants, such as geraniums. Additional preferred plants include
leafy green ornamental plants, such as Ficus, palms, and the
like, in which longevity of the leaf stem on the plant (delayed
abscission) is of particular relevance. Delayed flowering in
such plants may also be advantageous. Similarly, other preferred
plants include fruiting plants, such as banana and orange,
wherein pectin-dissolving enzymes are involved in the abscission
process.
The present invention will benefit plants subjected to
stress. Stress includes, and is not limited to, infection as a
result of pathogens such as bacteria, viruses, fungi, and
conditions involving aging, wound healing and soil penetration.
Bacterial infections include, and are not limited to,
Clavibacter michiganense (formerly Coynebacterium michiganense),
Pseudomonas solanacearum and Erwinia stewartii, and more
particularly, Xanthomonas campestris (specifically pathovars
campestris and vesicatoria), Pseudomonas syringae (specifically
pathovars tomato, maculicola).
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In addition to bacterial infections, other examples of
plant viral and fungal pathogens within the scope of the
invention include, but are not limited to, tobacco mosaic virus,
cauliflower mosaic virus, turnip crinkle virus, turnip yellow
mosaic virus; fungi including Phytophthora infestans, Peronospora
parasitica, Rhizoctonia solani, Botrytis cinerea, Phoma lingam
(heptosphaeria maculans), and Albugo candida.
In Arabidopsis, several expressed sequence tags with
significant similarity to the functional equivalent of yeast
Atxlp have been identified (Himelblau et al., Plant Physiol.
117:1227-1234 (1998)), indicating that other steps in
intracellular trafficking copper ions may also be conserved in
the plant kingdom. Although mutations have not been identified
in any of these genes, one or more of these proteins may also be
expected play a role in the creation of functional ethylene
receptors and would be known to one of ordinary skill in the art
in view of the findings of the present invention. Through
continued isolation of mutants using the Ran-/Ctr- screen of the
present invention (in particular from segregating families to
enable recovery of lethal mutations), it will be possible, in
accordance with the present invention, to identify additional
genes required for assembly of functional ethylene receptors.
The present invention is further described in the following
examples. These examples are not to be construed as limiting the
scope of the appended claims.
EXAMPLES
Example 1: Screen for Mutants Responsive to Potent
EthyleneReceptor Antagonist.
Arabidopsis thaliana ecotype Columbia (Col-0) was used as
the parental strain for mutant isolation. EMS (ethyl methane
sulfonate) mutagenesis of seeds was performed as described by
Guzman and Ecker, 1990, supra. Plant growth in hydrocarbon-free
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air, ethylene and TCO were carried out as described by Kieber et
al., 1993, supra.
To identify novel components required for ethylene
perception/signaling, mutants were screened that displayed
hormone responsive phenotypes upon exposure to an antagonist of
ethylene action. Trans-cyclooctene (TCO) was chosen as the
ethylene antagonist for the screen because this cyclic olefin
acts as a potent competitive inhibitor of ethylene binding to its
receptors) in vitro and in vivo (Sisler, 1990 supra; Schaller
et al., 1995, supra).
The screening of 30,000 M2-mutagenized seed populations,
which were selected from twenty (20) independent lots of seeds
that had been mutagenized with EMS, yielded two independent
responsive-to-antagonist) (rant) mutants that displayed a
characteristic "ethylene" triple response phenotype in response
to treatment with TCO. Complementation tests revealed that these
two mutants (ran)-1 and ran)-2) were allelic (see Table 1).
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Table 1- Genetic analysis of ranl mutants
Treatment Segregation Rati
Crossing o
ranl-1 x wild F1 TCO all wild type
type
F2 TCO ran-a; 94, wild type; 1:3 0.68
152
ranl-2 x wild F1 TCO all wild type
type
F2 TC0 ran-; 28, wild type; 1:3 5.32
135
ranl-I x ranl-2 F1 TCO all ran-
F2 TCO all ran'
ranl-1 x etrl-1 F1 TCO ran-; 8, wild type; 1:15 0.067
109
F2 ACC ein-; 151, wild type; 3:1 1.88
62
ranl-2 x etrl-1 F1 TCO ran-; 16, wild type; 1:15 4.62
137
F2 ACC ein-; 195, wild type; 3:1 0.29
60
ranl-2 x etrl-1 F1 TCO ran-: 15, wild type 1:15 0.0025
228
F2 ACC ein-; 143, wild type; 3:1 1.60
58
ranl-2 x ein2-12 F1 TCO ran-; 25, wild type; 1:15 6.31
222
F2 ACC ein-; 193, wild type; 3:1 1.60
58
a: Ran' indicates plants that display ethylene response
phenotypes when treated with TCO.
b: Ein- indicates plants that show absence of ethylene
response phenotypes.
Crosses were performed as described by Guzman and Ecker,
1990, supra. Fl progeny from crosses between wild-type plants
and ranl-I or rant-2 did not display the triple response
phenotype in the presence of TCO, while F2 progeny from self-
fertilized F1 plants showed a segregation ratio of 1 ran . 3
wild-type plants (see Table 1). This indicated that ranl is a
single locus recessive mutant.
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Example 2: Ethylene Response Pathway Is Activated in
ranl Mutants by an Ethylene Antagonist.
As previously shown, TCO treatment causes reversion of the
triple response phenotype of etol, a mutant that overproduces
ethylene, but not the phenotype of cntl, a mutant in which the
ethylene signaling pathway downstream of the receptors is
constitutively active. Although the ranl mutant seedlings
exhibit the triple response-like phenotype in the presence of
TCO, the same response is not seen when the seedlings are grown
in hydrocarbon-free air (Fig. 2A).
Effect of Mutation in Presence of Ethylene
Agonist on Seedlings
In ranl-1 and ranl-2, exposure to TCO produced effects on
seedling growth similar to those evoked by treatment of wild type
seedlings with ethylene (Fig. 2A). Measurements of the hypocotyl
and root length confirmed these observations. In the presence
of TCO, the length of the hypocotyl in ranl-1 and ranl-2
seedlings was 3.8 ~ 0.7 mm and 4:3 ~ 0.5 mm, respectively. In
ethylene-treated wild-type seedlings, the length of the hypocotyl
was 4.2 ~ 0.4 mm. Similarly, TCO-treatment caused inhibition of
root growth in ranl seedlings; although the length in ranl
mutants (ranl-1; 1.9 ~ 0.6 mm, ranl-2; 2.4 ~ 0.4 mm) was slightly
longer than of wild type in ethylene (1.2 ~ 0.2 mm).
The degree of curvature of the hypocotyl hook was also
measured in wild-type and mutant seedlings. In ethylene, the
angle of the hypocotyl hook in wild-type seedlings (250 ~ 49.2 )
was indistinguishable from that observed in TCO-treated ranl
mutants (ranl-1~ 250 ~ 23.7 , ranl-2; 253 ~ 23.8 ), further
demonstrating that TCO activates the ethylene signaling pathway
in ranl.
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S In contrast to its effect on the ranl mutants, TCO
treatment of wild-type seedlings caused an increase in hypocotyl
and root growth, compared with growth in hydrocarbon-free air
(Fig. 2A). These results are consistent with previous findings
that inhibition of basal level ethylene biosynthesis/perception
allows maximal elongation of seedlings (Guzman & Ecker, 1990,
s upra ) .
Other compounds that inhibit ethylene action, such as
silver ion or 1-methylcyclopropane (MCP) (Sisler et al., Plant
Growth Regul. 18:79-86 (1996)), failed to induce the triple
response in the ranl mutants. As in wild-type plants, these
compounds prevented responsiveness to ethylene in ranl mutants,
demonstrating that the ranl phenotype was specific to TCO.
Induction of the seedling triple response in rant mutants was
stereo-specific. Trans- but not cis-cyclooctene was effective
in evoking this morphological transformation. Furthermore,
treatment of ranl seedlings with TCO in the presence of AVG
(aminoethoxyvinylglycine), a potent inhibitor of ethylene
biosynthesis in Arabidopsis seedlings (Guzman & Ecker, 1990,
supra) , had no effect on the ranl mutant phenotype (Fig. 2A) ,
further demonstrating that the seedling phenotype typifies the
response to TCO ethylene antagonist, and that it was not due to
an increase in ethylene biosynthesis.
Induction of the triple response was examined in ranl
plants to different doses of ethylene. However, there appeared
to be no significant difference between mutant and wild-type
plants, suggesting that the rant mutations do not alter normal
perception of ethylene. When grown in hydrocarbon-free air, the
phenotype of ranl-2 seedlings was identical to the wild-type
phenotype (Fig. 2A). However, the root length of ranl-1 was
slightly shorter than that observed in wild-type seedlings.
Although these lines were extensively backcrossed, it was
possible that the short root phenotype of ranl-1 was caused by
a mutation near the RAN1 locus. However, since ranl-2 did not
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display this phenotype, it was concluded that this effect was
allele specific. No other significant growth phenotypes were
observed in ranl-I or ranl-2 seedlings or adult plants.
Effect of Mutation in Presence of Ethylene
Agonist on Adult Plants
For analysis of adult plants, seedlings were moved from
agar plates containing MS medium (Mirashugi and Skoog medium) to
soil, or they were germinated directly on soil, and young rosette
plants (after the second true. leaves have emerged) were exposed
to hydrocarbon-free air or TCO in 5-liter vessels for twelve (12)
days. The vessels were flushed daily with hydrocarbon-free air
and supplied with fresh TCO to reduce the accumulation of CO2, an
inhibitor of ethylene action.
To examine the effect of TCO on vegetative growth, young
rosette plants were exposed to TCO. The size of the leaves in
mutants of ranl-1 and ranl-2 was found to be significantly
reduced, as compared to wild type (compare Fig. 2A with Fig.
2B). Leaf shape and petiole length observed in TCO-treated rant
mutants phenocopied the morphology of ctrl-1 and were
characteristic of ethylene effects on plant growth (Chao et al.,
1997, supra).
The size of the leaves of the wild-type plants was also
slightly reduced by treatment with TCO (Fig. 2B). However, this
effect was assumed to be caused by the toxicity evoked by
treatment with a high level of TCO since a similar size reduction
was also observed in the constitutive ethylene signaling mutant
ctrl-1 plants. Measurement under a microscope of epidermal cells
of leaves from wild-type and ranl plants indicated that the
overall reduction in the size of TCO treated ranl leaves was due
to a decrease in cell elongation. Consequently, the rant
mutations and RAN1 gene expression products were shown to affect
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not only growth of seedlings, but also growth in adult plants,
and it was concluded that in rant mutants the ethylene receptors
have altered or relaxed ligand specificity.
Effect of Ethylene Agonist on Ethylene-Inducible Genes
To determine the effect of an ethylene antagonist on ranl
plants at the molecular level, several well-known ethylene-
inducible genes were treated with TCO and examined. EI305 (Chao
et al., 1997, supra) and GST2 (Itzhaki et al., Plant Mol. Biol.
22:43-58 (1993)) expression were examined by northern blot
analysis using total RNA from seedlings treated with ethylene or
TCO (Fig. 3A). As expected in wild-type plants, the steady state
mRNA levels for these two hormone response genes increased upon
treatment with ethylene, and decreased to below basal levels by
TCO treatment. Exposure to TCO significantly reduced EI305 and
GST2 expression in etol-5, whereas their expression was
unaffected in the ctrl-1 mutant (Fig. 3A), confirming that TCO
acts as an inhibitor of ethylene action.
In rant mutants, the steady state levels of EI305 and GST2
mRNAs were not inhibited by TCO, as observed in wild-type
seedlings. In fact, consistent with the agonist-like effects of
TCO on seedling morphology in rant plants, TCO caused a slight,
but reproducible, increase in the steady state mRNA levels of the
two genes (Fig. 3A).
Effect of Mutation on Agonist Response in
Late Stage Development
The effect of the ranl mutation on TCO responsiveness in
later stages of development was also examined. Basic-chitinase
(CHIB) gene expression is known to be up-regulated by ethylene
in adult Arabidopsis plants (Samac et al., Plant Phys.iol. 93:907-
914 (1990)) and dependent upon an intact ethylene signaling
pathway (Chen & Bleecker, Plant Physiol. 108:597-607 (1995)).
Northern blot analysis showed that the CHIB gene expression was
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induced in ranl mutants by exposure of rosette plants to TCO
(Fig. 3B). Taken together, these results demonstrated that in
rant seedlings and adult plants, TCO mimics the action of
ethylene in both the morphological and molecular aspects of the
response.
Expression of the ethylene inducible genes was further
examined in rosette plants. The basic chitinase gene (CHIB) and
one of the defensin genes (PDF1.2) are known to be induced by
ethylene treatment [Samac et al., 1990, supra; Penninckx et al.,
1996, supra]. However, northern blot analysis showed that the
expression of those genes were induced by one (1) day of TCO
treatment in ranl rosette plants (Fig. 3B), further confirming
that TCO activates the ethylene signaling pathway in rant mutants
in both seedlings and adults plants.
Example 3: RAN1 Acts Early in the Ethylene Gas
Signaling Pathway.
To determine where RAN1 acts in the ethylene signaling
pathway, epistasis analyses were performed using ranl and the
ethylene insensitive mutants, etrl and ein2. In the presence of
TCO, F2 progenies derived from self-fertilized etrl-1/+, ranl-1/+
or ein2-12/+, ranl-1/+ F1 plants displayed a phenotypic
segregation ratio of 1 Ran- to 15 wild-type plants. On the other
hand, the F2 progeny. segregated ein . wild type in ethylene,
demonstrating a ratio of 3 Ein- to 1 wild-type plants . Given
that etrl-1 is dominant (Bleecker et al., 1988, supra) and ein2-
I2 is semi-dominant (J. M. Alonzo and J.R. Ecker, unpublished
results), it became apparent that etrl-I and ein2-12 masked the
ranl mutant phenotype.
Using a PCR-based allele assay for ranl-1, homozygous ranl
mutants were identified among the F2 progenies derived from
crosses of ranl-1 to etrl-1 and ein2-I2. The phenotypes of both
ranl-l, etrl-1 and ranl-1 ein2-12 double mutants were Ein-, Ran+,
an
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confirming that etrl and ein2 are epistatic to ranl. Since ETR1
encodes an ethylene receptor, these genetic studies demonstrated
that RAN1 function is required very early in the hormone-
signaling pathway.
Furthermore, etrl-1 and ein2-12 were clearly ethylene
insensitive, further supporting the conclusion that TCO treatment
activates the ethylene signaling pathway in ranl mutants. After
determining the mutation site of ranl-1, it was possible to
confirm these results at the molecular level by finding several
ranl-1 homozygotes in ein seedlings in F2 progeny from both ranl
1 x etrl-I and ranl-1 x ein2-12 crosses.
Example 4: RANT Gene Encodes a Copper Transporting
P-type ATPase.
The ranl-1 mutant gene was mapped with visible markers by
crossing it with the W13 and W100 lines (from the Arabidopsis
Biological Resource Center at Ohio State University) and
examining segregation of the rant and ttg, yi, tt3, markers.
SSLP markers, Ath0109 and nga129, were used. (Bell & Ecker,
Genomics 19:137-144 (1994)). Newly synthesized SSLPs, (simple
sequence length polymorphisms) smMLNl and smMCLl9, a cleaved-
amplified polymorphic sequence (CAPS) CIC4E12R and a derived
cleaved-amplified polymorphic sequence (dCAPS) CIC2A12L were used
for mapping ranl-1.
DNA samples for PCR (polymerase chain reaction) based
marker analysis were isolated with CTAB (cetrimethyl-
amonimumbromide) methods. Briefly, 3-4 young leaves from
individual F2 progeny were frozen in liquid nitrogen and ground
with vigorous shaking in a Capmix (ESPE, America, Inc., USA) with
glass beads. 500 liters of the CTAB solution (100 mM Tris-HC1
pH 8.0, 20 mM EDTA pH8.0, 1.4 M NaCl, 3~ w/v CTAB) were added,
then heated at 65 C for 30 min. After phenol/chloroform
extraction, nucleic acids were precipitated by adding equal
volume of iso-propanol and resuspended in 100 ul of TE buffer (10
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S mM Tris-HCI, 1mM EDTA, pH 8.0). RNase was added to a
concentration of 10 g/1, the sample was incubated for 30 min at
37 C, followed by phenol/chloroform extraction and ethanol
precipitation. Precipitated nucleic acids were resuspended in
50 ul of TE buffer. PCR reactions (10 mM Tris-HC1 pH9.0, 50 mM
KC1, 2.5 mM MgClZ, 0.2 mM dNTPs, 5 pM each primers, 0.1 ~ Triton
X-100, 1 ul of nucleic acid sample, Taq polymerase, total volume
ul, were passed through 35 cycles of 30 seconds at 94 C, 30
seconds at 56 C, 30 seconds at 72 C.
To determine the nucleotide sequence of the RANI gene in
15 wild-type plants. and ranl mutants, synthetic oligonucleotide
primers were made (20-25bp, >50~ GC) that would enable sequencing
of the entire gene by primer walking. Genomic DNA was prepared
from the leaves of wild-type plants, ranl-1 and ranl-2 mutants.
Using synthetic primers, four overlapping DNA fragments were
amplified by PCR and sequenced after purification through agarose
gel electrophoresis. To avoid errors due to PCR, more than four
independent PCR samples were mixed and batch sequenced.
Synthetic oligonucleotides (5'-3' direction) used for
genetic marker production were as follows:
smMLNl-a; 5'-GTGGGTTGTTTCCGGCTAAG-3' (SEQ ID N0:4)
smMLNl-b; 5'-GCCAGTCACCAGAACCAGC-3' (SEQ ID N0:5)
smMCPl9-a; 5'-TACCATAGTGTCCTTCAACGG-3' (SEQ ID NO:&)
smMCPl9-b; 5'-TGGACCTGTAATCGGAGACG-3' (SEQ ID N0:7)
T19K24-la; 5'-CTTGGTGATCGAACCACGAAGGGAC-3' (SEQ ID N0:8)
T19K24-lb; 5'-CAAAGGACTCAATCTCAACCTACGC-3' (SEQ ID N0:9)
T19K24-3a; 5'-TTTCAGTTATACGTGGTTAATTTCGC-3' (SEQ ID N0:10)
T19K24-3b; 5'-GTCATGAGATTATGAGGTGCCGAC-3' (SEQ ID N0:11)
T19K24-4a; 5'-GCTATTCATCAGTCATCATCCCC-3' (SEQ ID N0:12)
T19K24-4b; 5'-GCGATAATTCTAGCATCCGATGC-3' (SEQ ID N0:13)
CAPS CIC4E12R-a; 5'-AATGAAACACGCTCATGTGCTCACC-3' (SEQ ID N0:14)
CAPS CIC4E12R-b; 5'-TCTCGCAGGAGTTTCCCTTTGAGCC-3' (SEQ ID N0:15)
dCAPS CIC2A12L-a; 5'-AGGACGTGATTGCTTGTGTAGGAGG-3' (SEQ ID N0:16)
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dCAPS CIC2A12L-b (mut); 5'-CAGTTTCTGGTTCAAGGAATAACTT-3' (SEQ ID
N0:17)
The amplified DNA produced using the CAPS primers was
subjected to MboII digestion; whereas the amplified DNA produced
using the dCAPS primers was subjected to Xmnl digestion.
For epistasis analyses, ranl mutants and etrl-1 or ein2-12
were crossed and progeny of individual F1 plants were collected.
The F2 seeds were germinated in the presence of ACC (1
aminocyclopropane-1-carboxylic acid) or TCO, then the Ein
seedlings in the presence of ACC and Ran- seedlings in TCO were
counted. The ranl-1 alleles were distinguished from wild type
by digestion of PCR fragments with BsrI; the ranl-1 mutation
eliminates a BsrI restriction site. The ranl-1, etrl-1 and ranl-
1, ein2-12 double mutants were confirmed at molecular level by
identifying ranl-1 homozygotes in Ein' seedlings of F2 progeny
from both rant-1 x etrl-1 and ranl-I x ein2-12 crosses using the
ranl-1 polymorphism.
Chromosomal and Fine MappincL of ranl
The ranl-1 mutation mapped close to the tt3 gene on
chromosome 5, then fine mapping was done using SSLP markers.
Based on the analysis of 2018 recombinant chromosomes derived
from a cross between ranl-1 and the Landsberg strain (Ler), the
position of ranl was narrowed to a region near the CRAI locus
(Fig. 4). Ler was from the Arabidopsis Biological Resource
Center at Ohio State University.
Development of additional PCR-based genetic markers (smMLN1
and smMCPl9) and their use in assembly of a BAC contig, permitted
the localization of ranl to an interval 1.8 cM from smMLNl and
2.6 cM from smMCPl9 (Fig. 4). To further delineate the location
of ranl, a CAPS marker, CIC2A12 (Konieczny et al., Plant J.
4:403-410 (1993)) was developed from the right-end of YAC (yeast
artificial chromosome) CIC4E12 and a dCAPS sequence, CIC2A12
(Michaels et al., Plant J. 1Q:381-385 (1998); Neff et al., Plant
d3
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J. 14:387-392 (1998)) was produced from the left-end of YAC
CIC2A12. Using the two genetic markers, several additional BAC
clones were identified and a BAC contig spanning about 160 kb was
assembled that contained the RAN1 gene (Fig. 4).
Among the BACs, one clone unassigned chromosomal location
(T19K24; GenBank accession AC002342) was fully sequenced, and a
number of gene models were predicted. By identifying recombinant
events using two additional SSLP markers (T19K24-1 and T19K24-4)
derived from sequences near the BAC ends, the location of RAN1
on the BAC clone was confirmed (Fig. 4). By selecting from among
the numerous predicted open reading frames in the RANlregion of
BAC T19K24, a predicted protein with similarity to copper
transporting P-type ATPases was also identified. Because it has
been proposed that the binding of ethylene to its receptor
requires the presence of a transition metal, this gene was
selected as the candidate for RAN1.
Genomic Sequencing of ranl
Genomic DNA from the region was sequenced in ranl-1 and
ranl-2 and the parental strain (Col). Single base changes were
identified in both ranl alleles (see below) confirming that this
gene was, in fact, RAN/ (Fig. 5). The genomic sequence of RAN1
was recorded at GenBank with accession #AF091112 and is
reproduced herein as SEQ ID N0:1.
cDNA Seauencinq of ranl and Putative Amino Acid Sequence
Using the PCR fragments mentioned above, a cDNA library was
screened and several cDNAs were isolated. One cDNA (pNH633),
containing the full RAN1 coding region, was subjected to sequence
analysis. Sequencing from the 5' and 3' ends of a second RANT
cDNA was also performed to confirm the position of initiation and
stop codons. Total RNA extraction and northern analysis were
performed as described (Kieber et al., 1993, supra).
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Several cDNA clones were isolated from the ethylene-treated
etiolated seedling library. Determination of the nucleotide
sequence of the longest cDNA and comparison with the genomic
sequence revealed that the candidate RAN1 gene contained nine
exons and eight introns (Fig. 5), distinct from the computer
predicted gene model. The cDNA sequence of RAN1 was recorded at
GenBank with accession #AF082565 and is reproduced herein as SEQ
ID N0:2. The amino acid sequence of RANT was predicted from the
expressed sequences (exons) of the RAN1 cDNA to be a polypeptide
of 1001 amino acid residues, which is reproduced herein as SEQ
ID N0:3.
Comparing Amino Acid Sequence of RAN1 with Other Known
Copper Transporters
The amino acid sequence of RAN1 was aligned with other
copper transporting P-type ATPases from human (ascession # Menkes
Q04656; Wilson P35670) , C. elegans (ascession # D83665) and
budding yeast (ascession # P38995). These proteins are
localized to a post-Golgi compartment where they function to
transport copper ions into the secretory pathway, delivering
copper to secreted or membrane bound proteins that require this
metal for functionality (Petris et al., EMBO J. 15:6084-6095
(1996); Yamaguchi et al., Proc. Natl. Acad. Sci. USA 93:14030
14035 (1996); Yuan et al., J. Biol. Chem. 272:25787-25793
(1997)).
Significant portions of the polypeptides were found to have
identity and conservative changes, to the extent that putative
metal binding motifs and predicted functional domains were
determined, particularly to the human Menkes disease gene
product, ATP7A (Chelly et al., Nature Genetics 3:14-19 (1993);
Mercer et al., Nature Genetics 3:20-25 (1993); Vulpe et al.,
Nature Genetics 3:7-13 (1993)), the human Wilson disease gene
product, ATP7B (Bull et al. , Nature Genetics 5: 327-337 (1993) ;
Chelly & Monaco, Nature Genetics 5:317-318 (1993); Petrukhin et
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al., Nature Genetics 5:338-343 (1993).), a copper transporter of
Caenorhabditis elegans (Sambongi et al., J. of Biochem. (Tokyo)
121:1169-1175 (1997)) and yeast Ccc2p (Fu et al., Yeast 11:283-
292 (1995); Yuan et al., Proc. Natl. Acad. Sci. USA 92:2632-2636
(1995)). These proteins share several structural features,
including 1) amino-terminal metal-binding motifs, 2) a
phosphatase domain, 3) a transduction domain, 4) a
phosphorylation domain, and 5) an ATP binding domain. The RAN1
protein possesses all of these features in regions of high
similarity between RAN1 and other copper transporters.
Interestingly, RAN1 and Ccc2p have only two putative metal
binding motifs in their N-terminal regions, whereas Menkes and
Wilson proteins have six, and a predicted nematode gene has three
motifs. Like these proteins, the candidate RAN1 protein contains
eight putative membrane-spanning regions.
In the ranl-1 mutant allele, a C to T transition was found
at nucleotide 1880 (corresponding to genomic sequence with the
A from the first ATG codon assigned the number 1). This base
change causes an amino acid change from Thr 497 to Ile.
Threonine 497 is located in the phosphatase domain, and this
residue is conserved in all copper transporters (Fig. 5A). This
suggested that the amino acid change found in ranl-1 reduces RAN1
function.
In ranl-2, a G to A base change was found at nucleotide
637, changing Gly 173 to Glu. Glycine 173 is not highly
conserved among copper transporters, but the corresponding
residues in the metal binding repeats of copper transporters are
not charged amino acid residues (Fig. 5B). Recent analysis of
the three dimensional structure of the fourth metal binding
repeat of the Menkes protein indicated that the residue
corresponding to Gly 173 of RAN1 contributes to the hydrophobic
core that is required for the correct positioning of the metal
binding loop (Gitschier et al., Nature Structural Biology 5:47-54
(1998)). Thus, the conversion from Gly to Glu causes a dramatic
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change in charge and hydrophobicity, conceivably disturbing the
structure of the metal binding domain, which is necessary for
copper transporter function (Payne et al., J. Biol. Chem.
273:3765-3770 (1998) ) .
Neither of the two mutations, however, affected the
expression of the RAN1 gene, at least at the transcriptional
level, since RAN1 mRNA was detected in both mutant strains to
much the same extent as it was detected in wild type plants.
Example 5: Expression of RANT cDNA rescues the Fet3p
deficient phenotype of the yeast ccc2
disruption mutant.
Because of its extensive similarity to known copper
transporters, RAN1 was predicted to have copper transporting
activity. To confirm this hypothesis, yeast complementation
analyses were performed using a budding yeast ccc2 disruption
(ccc2a) mutant.
In budding yeast, Ccc2p delivers copper ions from the lumen
to the cytosol of the secretion pathway (Yuan et al., 1995,
supra). The copper binding domain of the multi-copper oxidase,
Fet3p, is loaded with copper, which is required for Fet3p oxidase
activity. Thus, the oxidase activity of Fet3p provides the basis
for an in vivo assay for copper transporter activity. In the
absence of Ccc2p, Fet3p has no oxidase activity. However, the
ccc2- defect can be rescued by homologous copper transporters
from other species (Hung et al., J. Biol. Chem. 272:21461-21466
(1997) : Payne et al., 1998, supra; Sambongi et al., 1997, supra) .
Consequently, to confirm that RAN1 was able to suppress the
Fet3p oxidase defect of ccc2~, recombinant plasmids were
constructed in which RAN1 cDNA was under the control of the
constitutive PGK1 promoter.
The following were used for the yeast complementation
analyses: 2908 (strain 7) [MAT(a), his3-200, leu2, trpl-101,
ura3-52, ade5] and ccc2(l1) (strain 8) [MAT (a) , his3-200, trpl-
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101, ura3-52, ade5,ccc2::LEU2]. For construction of plasmids
containing the ranl-1 mutation, two primers were designed:
ranl-la: 5' ATGAATCCATGGTGATTGGTGAATCAGTTCC-3' (SEQ ID
N0:18); and
ranl-lb: 5'-CAGTCACCATGGATTCATTAACGTAACTTG-3' (SEQ ID
NO: 19) .
The underlined T corresponds to the rant-1 mutation. Italic
characters indicate an introduced NcoI restriction site.
Using these two primers and two primers corresponding to
nucleotides 2632-2608 (#20) and nucleotides 934-958 (#7), two
fragments were amplified by PCR using the cloned RAN1 cDNA as
template. The ranl-la-#20 PCR fragment was digested with NcoI
and BspEI, while ranl-Ib-#7 PCR fragment was digested with NcoI
and StuI. The cDNA region from StuI to BspEI sites was exchanged
with ranl-la-#20 and ranl-lb-#7 PCR fragments.
For construction of plasmids containing the ranl-2
mutation, two primers were designed.
ranl-2a: 5'-AGCTTTGTCGACATCATTAGAAGAAGTTGAG-3' (SEQ ID N0: 20);
and
ranl-2b: 5'-AATGATGTCGAC~AAGCTACCACTGCTC-3'.(SEQ ID N0: 21).
The underlined A corresponds to the ranl-2 mutation.
Italic characters indicate an introduced SalI restriction site.
Using these two primers and another primer corresponding to
nucleotides 1439-1416 (#18) and the T7 primer for the sequence
of pBluescript, two fragments were amplified by PCR using the
cloned RAN1 cDNA as template. The ranl-2a-#18 PCR fragment was
digested with SalI and StuI, while the ranl-lb-T7 PCR fragment
was digested with SalI and ClaI. The Clal to StuI region was
exchanged with ranl-2a-#18 and rant-lb-T7 PCR fragments. Wild-
type and mutant RANI cDNAs were introduced into pYE53 (PGK1
promoter). Transformation of yeast with various plasmids was
performed using the lithium acetate method of Ito et al., J.
Bacteriol. 153:163-168 (1983).
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Yeast cells were grown in YPD (yeast, peptone and dextrose)
medium at 30°C to a density of 0.3 OD600. The cells were
harvested by centrifugation and washed with ice-cold extraction
buffer (25 mM Tris pH 7.5, 100 mM NaCl, 1 mM phenylmethylsulfonyl
fluoride, 10 mM leupeptin, 10 mM antipain). Yeast cells were
homogenized in the extraction buffer by mixing with glass~beads.
After removal of large particles, homogenates were centrifuged
(20,000 x g) at 4°C for 20 min. Pellets were washed in
extraction buffer, resuspended in the same buffer containing to
Triton X-100, and centrifuged again to precipitate undissolved
materials. Protein, samples (25 mg) were mixed with the same
volume of SDS page buffer (100 mM sodium phosphate, pH 7.2; 0.5~
SDS: 10~ (w/v) glycerol) and separated by SDS/PAGE without prior
heating.
After electrophoresis, the gel was equilibrated with 0.005
Triton X-100 in l00 (w/v) glycerol, and then soaked in 3 mM p-
phenylenediamine dihydrochloride (Sigma}, 100 mM sodium acetate,
pH 5.8, 1 mM NaN3. The gel was air-dried in the dark at room
temperature. To detect RAN1 protein in yeast, a triple repeat
of the HA-tag sequence was introduced into RAN3, ranl-1 and ranl-
2 cDNAs, just before the stop codon. Western blot analysis was
performed using monoclonal anti-HA antibody.
Transformants of the ccc2~ strain, in which the plasmid DNA
containing RAN1 cDNA, were found to have detectable Fet3p oxidase
activity, although the activity was somewhat less than that of
the wild type. By comparison, the same strain transformed with
vector plasmid DNA alone, did not have such activity (Fig. 6A).
In addition, copper transporter activity could be
determined by monitoring the iron uptake of yeast cells since
Fet3p has a pivotal role in high-affinity iron uptake in budding
yeast (Stearman et a1, Science 271:1552-1557 (1996)). The iron
uptake assay was performed as described by Dancis et al., J.
Biol. Chem. 269:25660-25667 (1994). Cells from the transformed
yeast strains were inoculated into 100 ul of YPD in the wells of
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a microtiter plate and grown at 30°C. After 14 hrs of
incubation, the cells were diluted 1:5 into fresh YPD medium in
a volume of 50 ul. After incubation for an additional 2 hrs, the
ssFe radionuclide (Amersham 37-50 mCi/mg iron) was added in 50 ul
of citrate buffer. The uptake was allowed to proceed for 2 hours
at which point the cell number was determined by measuring the
turbidity (OD720). The cells were then collected on glass fiber
filters and washed with water. The filters were counted in a
Beckman scintillation counter and the cell-associated
radioactivity was calculated. For preparation of the
radionuclide, 55Fe was prepared by reduction with sodium
ascorbate and diluted to a concentration of 2 uM in citrate
buffer (50mM sodium citrate pH 6.5, glucose 5~). The iron
preparation was then added to the cells in microtiter wells.
Without Ccc2 function, iron uptake of yeast cells was found
to be reduced. In the ccc2a cells expressing RAN1 cDNA, high
affinity iron uptake was restored relative to the negative
control cells, which carried only the empty vector, (Fig. 6B),
further demonstrating that RAN1 has copper transporter activity.
To confirm that the ranl mutant gene products have reduced
activity, the ranl-1 mutation and the ranl-2 mutation were
respectively introduced into wild type RAN1 cDNA. Then, ccc2a
was transformed with the plasmid containing those mutant cDNAs.
Both the ranl-1 and ranl-2 defects were found to impair copper
transporting activity, although notably the ccc2o transformants
harboring the ranl mutant cDNAs had a significantly reduced Fet3p
oxidase activity and high affinity iron uptake activity when
compared to RAN1. While transformants containing ranl-2 cDNA
also showed reduced activity, the level of Fet3p oxidase activity
and iron uptake activity was higher than that observed in
transformants expressing ranl-1. Western blot analysis revealed
that RAN1, rant-2 and ranl-2 proteins accumulated to the same
level in the yeast.
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One explanation for the somewhat greater activity of the
ranl-2 may be that in RAN1, which contains only a single
functional metal binding motif, there is sufficient copper
transporting activity in yeast, but not in plant cells. As shown
previously, expression of a Wilson's protein containing only a
single binding motif was sufficient for the functional
complementation of yeast ccc2 deletion mutant (Iida et al., FEBS
Lefts 428:281-285 (1998)).
Example 6: Presence of Copper Ions Suppresses the Ethylene
Phenotype of ranl Plants.
The addition of copper ions to the media suppresses the
defect in Fet3p activity of the ccc2 disrupted mutant (Fu et al.,
1995, supra: Yuan et al. , 1995, supra) . Since RANT encodes a
Ccc2p homologue, it was important to determine whether or not the
addition of copper ions to the plant growth media could suppress
the rant phenotype, analogous to the effect on the yeast ccc2
mutants. Consequently, various concentrations of CuS04 (from 0.1
uM to 100 uM) were added to the growth media to evaluate the
effect on the germination and growth of seedlings of Arabidopsis.
At concentrations of 1 uM and below, the CuS04 did not have an
apparent effect. By contrast, however, concentrations of 50-100
uM CuS04 significantly inhibited germination.
Interestingly, an intermediate CuS04 concentration of 10-25
uM, which did not have any obvious effects on germination and
seedling growth, partially suppressed the ranl phenotype of both
ranl-I and ranl-2 (Fig. 7). Specifically, the copper
supplemented growth media prevented the rant seedlings from
responding to TCO, as evidenced by the lack of the triple
response phenotype. However, CuS04 at this concentration did
not interfere with TCO-mediated inhibition of the triple response
phenotype of etol-5 or ctrl-I (Fig. 7). Thus, it is not likely
that CuS04 , as a compound per se, affects the triple response
phenotype or the downstream ethylene signaling pathways.
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Fet3p is also involved in high affinity iron uptake. The
human Fet3p homologue, ceruloplasmin, has been implicated in the
iron metabolism in human cells, and its activity is dependent on
the proper functioning of the Menkes/Wilson proteins. Thus, to
evaluate the possibility that the ranl phenotype could also have
l0 been caused by a deficiency in high affinity iron uptake in
Arabidopsis, iron was added to the plant media to determine
whether or not the rant phenotype was suppressed in TCO. Several
concentrations of ferric ammonium sulfate (from 30 uM to 1 mM)
and of FeEDTA (from 1 uM to 1 mM) were added to the seedling
growth media, but no effect could be detected on the ranl
phenotype in the presence of TCO. Therefore, it was concluded
that although the phenotype of the ranl mutants is caused by a
defect in copper metabolism, the phenotype is not related to a
similar defect in iron metabolism.
Example 7: Constitutive Activation of Ethylene Responses
in RAN1 Transgenic Plants
To further explore the function of RAN1, the effect of
ectopic gene expression on seedling development/hormone
responsiveness was examined. To transform the plants the RAN/
cDNA was inserted between the CaMV 35S promoter and the nopaline
synthase (NOS) terminator sequence of pROK2 vector plasmid
(Baulcombe et al., Nature 321:446-449 (1986)) yielding the
plasmid pNH634. Then pNH634, containing the full length RAN1
cDNA, was introduced into Agrobacterium C58 strain and the
resulting strains were used to transform ranl-1, ranl-2 and wild-
type plants by the vacuum infiltration method, according to
Bechtold et al . , CR Acad. Science (Paris) 316:1194-1199 ( 1993 ) ) .
Interestingly, independent of their different genetic
backgrounds, two distinct phenotypic classes of transformants
were observed. Approximately one-half of all T1-generation
transgenic lines displayed a constitutive ethylene response
phenotype (class 1, Ctr-), while the other half of transformants
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did not show an obvious growth phenotype (class 2, Ctr+) (Fig.
8A). When treated with TCO, class 2 transformants were no longer
able to respond to the antagonist, indicating that the introduced
35S:RAN1 transgene had complemented the rant mutation. Northern
blot analysis revealed that class 2 transgenic plants expressed
a high level of RANI mRNA (Fig. 8B, upper panel). In contrast,
the level of RAN1 mRNA found in plants displaying a strong
constitutive ethylene response (Ctr-) phenotype was much reduced
(Fig. 8B, upper panel), an effect likely due to co-suppression
of the endogenous RAN1 gene.
The severe Ctr- phenotype observed in the RAN1 transgenic
plants suggested that activation of the ethylene-signaling
pathway was occurring. To examine this hypothesis, the
expression of the ethylene-responsive basic-chitinase (CHIB) gene
was examined in the 35S::RAN1 transformed plants (classes 1 and
2). Northern blot analysis of mRNA from plants that displayed
a Ctr- phenotype (class 1) revealed a significantly elevated
level of expression of basic-chitinase compared to expression in
untransformed plants (Fig. 7B, lower panel). By comparison,
phenotypically wild-type plants (class 2) showed only basal level
expression of basic-chitinase mRNA. These results confirm the
association between reduced RAN1 expression, the Ctr- phenotype
and activation of ethylene responsive gene expression in the
class 1 CaMV35S:RAN1 transgenic lines.
Because it was not possible to carry out further genetic
characterization of the class 1 plants due to complete
infertility in all lines, subsequent generations of class 2
plants segregating for the 35S::RAN1 transgene were studied. T2
generation plants from twenty-four (24} independent transformants
were permitted to self-fertilize, and the progeny were examined
for ethylene phenotypes. Seedlings from two 35S::RANI lines (L3
and L6) when grown in hydrocarbon-free air, displayed a
constitutive triple response phenotype identical to ctrl mutants
(Fig. 8C).
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In addition, treatment of L3 and L6 seedlings with ethylene
action inhibitors, silver ion or MCP (1-methylcyclopropane), had
no effect on the triple response phenotype, indicating that
activation of the ethylene response in these lines was not due
to overproduction of the hormone. Moreover, after several weeks
of growth, the resulting plants developed an adult morphology
identical to that observed in RAN1 co-suppressed plants (class
1). These studies revealed that a reduction of RANI expression
results in constitutive activation of ethylene morphology in both
seedlings and adult plants.
While the foregoing specification has been described with
regard to certain preferred embodiments, and many details have
been set forth for the purpose of illustration, it will be
apparent to those skilled in the art that the invention may be
subject to various modifications and additional embodiments, and
that certain of the details described herein can be varied
considerably without departing from the basic principles of the
invention. Such modifications and additional embodiments are
also intended to fall within the scope of the appended claims.
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