U.S. patent application number 09/729821 was filed with the patent office on 2002-06-06 for transgenic plants that exhibit enhanced nitrogen assimilation.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Kida, Takao, Kisaka, Hiroaki.
Application Number | 20020069430 09/729821 |
Document ID | / |
Family ID | 24932772 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020069430 |
Kind Code |
A1 |
Kisaka, Hiroaki ; et
al. |
June 6, 2002 |
Transgenic plants that exhibit enhanced nitrogen assimilation
Abstract
Transgenic plants containing free amino acids, particularly at
least one amino acid selected from among glutamic acid, asparagine,
aspartic acid, serine, threonine, alanine and histidine accumulated
in a large amount, in edible parts thereof, and a method of
producing them are provided. In this method, glutamate
dehydrogenase (GDH) gene is introduced into a plant together with a
regulator sequence suitable for over expressing the sequence
encoding GDH gene in plant cells.
Inventors: |
Kisaka, Hiroaki;
(Kawasaki-Shi, JP) ; Kida, Takao; (Kawaski-Shi,
JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
15-1, Kyobashi 1-chome
Tokyo
JP
|
Family ID: |
24932772 |
Appl. No.: |
09/729821 |
Filed: |
December 6, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09729821 |
Dec 6, 2000 |
|
|
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08132334 |
Oct 6, 1993 |
|
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Current U.S.
Class: |
800/290 ;
435/69.1; 800/278 |
Current CPC
Class: |
C12Y 104/01002 20130101;
C12Y 104/01004 20130101; Y02A 40/146 20180101; C12N 9/0004
20130101; C12N 15/8241 20130101; C12N 15/8261 20130101; C12N 9/0016
20130101; C12N 9/93 20130101; C12N 15/8251 20130101 |
Class at
Publication: |
800/290 ;
800/278; 435/69.1 |
International
Class: |
C12N 015/82; A01H
005/00; C12N 015/82 |
Goverment Interests
[0002] This invention was made with government support under grant
no.:GM32877 awarded by the National Institute of Health, and grant
nos.:DEFG0292 and ER20071 awarded by the Department of Energy. The
government has certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 1999 |
JP |
11-376710 |
Claims
What is claimed is:
1. A method of producing a plant with an improved agronomic or
nutritional characteristic by engineering the plant for ectopic
overexpression of one or several nitrogen assimilation/metabolism
enzymes, wherein a plant with an improved agronomic or nutritional
characteristic exhibits: i) faster rate of growth, ii) greater
fresh or dry weight at maturation, iii) greater fruit or seed
yield, iv) greater total plant nitrogen content, v) greater fruit
or seed nitrogen content, vi) greater free amino acid content in
the whole plant, or vii) greater free amino acid content in the
fruit or seed, viii) greater protein content in seed or fruit,
viiii) greater protein content in a vegetative tissue, than an
identically cultivated unengineered, progenitor plant, when said
plant and said progenitor plant are cultivated under nitrogen
non-limiting growth conditions; the overexpressed nitrogen
assimilation/metabolism enzyme is an aspartate aminotransferase,
glutamate 2-oxoglutarate aminotransferase, glutamate dehydrogenase,
asparaginase, eukaryotic asparagine synthetase or cytosolic
glutamine synthetase; and the engineering for ectopic
overexpression of one or several nitrogen assimilation/metabolism
enzymes comprises: i) transforming the plant with one or several
gene fusions that confer ectopic overexpression of one or several
of said nitrogen assimilation/metabolism enzymes, ii) selecting or
identifying the transformed plant based on the trait conferred by a
marker gene linked to said gene fusion, iii) screening the
transformed plant for one or more of above said improved agronomic
or nutritional characteristics when said transformed plant is
cultivated under nitrogen non-limiting growth conditions, iv)
selecting the transformed plant with one or more improved agronomic
or nutritional characteristic.
2. The method of claim 1, wherein the gene fusion comprises a gene
encoding a nitrogen assimilation/metabolism enzyme operably linked
to a strong, constitutively expressed plant promoter.
3. The method of claim 2, wherein said strong, constitutively
expressed plant promoter is a CaMV 35S promoter.
4. The method of claim 3, wherein the nitrogen
assimilation/metabolism enzyme is an eukaryotic asparagine
synthetase or cytosolic glutamine synthetase.
5. The method of claim 4, wherein the nitrogen
assimilation/metabolism enzyme is a root-specific glutamine
synthetase.
6. The method of claim 1, wherein the gene fusion is the 35S-GS
gene fusion of pZ3, pZ9, or pZ17.
7. The method of claim 1, wherein the gene fusion is the 35S-AS
gene fusion of pZ127.
8. A plant produced by the method of any of claims 1 to 7.
9. A seed of a plant produced by the method of any of claims 1 to
7, wherein said seed contains one or several of said gene fusions
that confer ectopic overexpression of one or several of said
nitrogen assimilation/metabolism enzymes.
10. A plant of the seed of claim 9.
11. A method of producing a plant with a suppressed level of
glutamine synthetase by engineering the plant for ectopic
overexpression of a glutamine synthetase gene, wherein the
suppressed level of glutamine synthetase is in comparison with
identically cultivated unengineered, progenitor plant; and the
engineering of the plant comprises: i) transforming the plant with
a gene fusion designed to confer ectopic overexpression of a
glutamine synthetase gene, ii) selecting or identifying the
transformed plant based on the trait conferred by a marker gene
linked to said gene fusion, iii) screening the transformed plant
for an abnormally low level of glutamine synthetase, and iv)
selecting the transformed plant with an abnormally low level of
glutamine synthetase.
12. The method of claim 11, wherein said glutamine synthetase gene
is a gene encoding chloroplastic glutamine synthetase.
13. The method of claim 12, wherein the gene fusion is the 35S-GS
fusion of pZ41 or pZ54.
14. A method of producing a plant with a suppressed level of
asparagine synthetase by engineering the plant for ectopic
overexpression of an inactive asparagine synthetase, wherein the
suppressed level of asparagine synthetase is in comparison with
identically cultivated unengineered, progenitor plant; and the
engineering of the plant comprises i) transforming the plant with a
gene fusion that confers ectopic overexpression of an inactive
asparagine synthetase, ii) selecting or identifying the transformed
plant based on the trait conferred by a marker gene linked to said
gene fusion, iii) screening the transformed plant for an abnormally
low level of asparagine synthetase, and iv) selecting the
transformed plant with an abnormally low level of asparagine
synthetase.
15. The method of claim 14, wherein the gene fusion is the 35S-AS
fusion of pZ167.
16. A progeny, clone, cell line or cell of a plant produced by the
method of any of claims 1 to 7, wherein said progeny, clone, cell
line or cell contains one or several of said gene fusions that
confer ectopic overexpression of one or several of said nitrogen
assimilation/metabolism enzymes.
17. A genetically engineered plant which (a) ectopically
overexpresses a gene encoding an aspartate aminotransferase,
glutamate 2-oxoglutarate aminotransferase, glutamate dehydrogenase,
asparaginase, eukaryotic asparagine synthetase or cytosolic
glutamine synthetase, and (b) exhibits one or more of the following
improved agronomic or nutritional characteristics: i) faster rate
of growth, ii) greater fresh or dry weight at maturation, iii)
greater fruit or seed yield, iv) greater total plant nitrogen
content, v) greater fruit or seed nitrogen content, vi) greater
free amino acid content in the whole plant, or vii) greater free
amino acid content in the fruit or seed, viii) greater protein
content in seed or fruit, viiii) greater protein content in a
vegetative tissue, than an identically cultivated unengineered,
progenitor plant, when said plant and said progenitor plant are
cultivated under nitrogen non-limiting growth conditions.
18. The genetically engineered plant of claim 17, wherein the
cytosolic glutamine synthetase is a root-specific glutamine
synthetase.
Description
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 08/132,334 filed Oct. 6, 1993, which is hereby
incorporated by reference in its entirety.
1. INTRODUCTION
[0003] The present invention relates generally to
genetic-engineering plants to display enhanced nitrogen
assimilatory and utilization capacities, grow larger, more
efficiently or rapidly, and/or have enriched nitrogen contents in
vegetative and/or reproductive plant parts and/or increased
biomass. More particularly, this invention relates to producing
transgenic plants engineered to have altered expression of key
enzymes in the nitrogen assimilation and utilization pathways. The
engineered plants may be productively cultivated under conditions
of low nitrogen fertilizer input or in nitrogen poor soils.
Alternatively, the engineered plants may be used to achieve faster
growing or maturing crops, higher crop yields and/or more
nutritious products under ideal cultivation conditions.
2. BACKGROUND OF THE INVENTION
[0004] Nitrogen is often the rate-limiting element in plant growth
and all field crops have a fundamental dependence on inorganic
nitrogenous fertilizer. Since fertilizer is rapidly depleted from
most soil types, it must be supplied to growing crops two or three
times during the growing season. Nitrogenous fertilizer, which is
usually supplied as ammonium nitrate, potassium nitrate, or urea,
typically accounts for 40% of the costs associated with crops such
as corn and wheat. It has been estimated that approximately 11
million tons of nitrogenous fertilizer is used in both North
America and Western Europe annually, costing farmers $2.2 billion
each year (Sheldrick, 1987, World Nitrogen Survey, Technical Paper
no. 59, Washington, D.C.). Furthermore, World Bank projections
suggest that annual nitrogen fertilizer demand worldwide will
increase from around 90 million tons to well over 130 million tons
over the next ten years. Increased use efficiency of nitrogen by
plants should enable crops to be cultivated with lower fertilizer
input, or alternatively on soils of poorer quality and would
therefore have significant economic impact in both developed and
developing agricultural systems.
[0005] Using conventional selection techniques plant breeders have
attempted to improve nitrogen use efficiency by exploiting the
variation available in natural populations of corn, wheat, rice and
other crop species. There are, however, considerable difficulties
associated with the screening of extensive populations in
conventional breeding programs for traits which are difficult to
assess under field conditions, and such selection strategies have
been largely unsuccessful.
[0006] 2.1. Nitrogen Assimilatory Pathway in Plants
[0007] Plants obtain nitrogen from their environment in the form of
inorganic compounds, namely nitrate and ammonia taken up from
roots, and atmospheric N.sub.2 reduced to ammonia in
nitrogen-fixing root nodules. Although some nitrate and ammonia can
be detected in the transporting vessels (xylem and phloem), the
majority of nitrogen is first assimilated into organic form (e.g.,
amino acids) which are then transported within the plant.
[0008] The first step in the assimilation of inorganic nitrogen
into organic form predominately involves the incorporation of
ammonia with glutamate to form glutamine, catalyzed by the enzyme,
glutamine synthetase (GS; EC 6.3.1.2). Glutamine thus formed may in
turn donate its amide group in the formation of asparagine,
catalyzed by the enzyme, asparagine synthetase (AS; E.C. 6.3.5.4).
The steady flow of nitrogen from ammonia to asparagine in this
pathway depends upon the recycling of glutamate and
.alpha.-ketoglutarate and aspartate, catalyzed by glutamine
2:oxoglutarate aminotransferase (GOGAT; E.C.) and aspartate
aminotransferase (AspAT; E.C.), respectively (see FIG. 1). Thus,
GS, AS, AspAT and GOGAT comprise the key enzymes of the main
nitrogen assimilatory pathway of higher plants.
[0009] Evidence exists indicating that ammonia incorporation may
proceed through alternative pathways other than that catalyzed by
GS (FIG. 1). See Knight and Langston-Unkefer, 1988, Science
241:951-954. One pathway may involve the incorporation of ammonia
with .alpha.-ketoglutarate to form glutamate, catalyzed by
glutamate dehydrogenase (GDH). Another pathway may involve the
incorporation of ammonia with aspartate to form asparagine,
catalyzed by asparagine synthetase (Oaks and Ross, 1984, Can. J.
Bot. 62:68-73; Stulen and Oaks, 1977, Plant Physiol. 60:680-683).
Since both of these enzymes (GDH and AS) have a high Km for
ammonia, the roles of these alternative nitrogen assimilation
pathways under normal growth conditions (e.g., low concentrations
of, internal ammonia) remain unclear. One study suggests these or
other alternative nitrogen assimilation pathways may make
significant contributions to a plant's nitrogen assimilation
capacity when intracellular ammonium concentration is elevated
above normal levels (Knight and Langston-Unkefer, id.).
[0010] 2.2. Nitrogen Transport and Utilization
[0011] Glutamine and asparagine represent the major long-distance
nitrogen transport compounds in plants and are abundant in phloem
sap. Aside from their common roles as nitrogen carriers, these two
amino acids have somewhat different roles in plant nitrogen
metabolism. Glutamine is the more metabolically active of the two
and can directly donate its amide nitrogen to a large number of
substrates in various anabolic reactions. Because of its
reactivity, glutamine is generally not used by plants to store
nitrogen.
[0012] By contrast, asparagine is a more efficient compound for
nitrogen transport and storage compared to glutamine because of its
higher N:C ratio. Furthermore, asparagine is also more stable than
glutamine and can accumulate to higher levels in vacuoles. Indeed,
in plants that have high nitrogen assimilatory capacities,
asparagine appears to play a dominant role in the transport and
metabolism of nitrogen. See Lea and Miflin, Transport and
metabolism of asparagine and other nitrogen compounds within the
plant, in The Biochemistry of Plants: A Comprehensive Treatise, vol
5. Amino acid and derivatives, Miflin ed., Academic Press, New York
(1980) pp 569-607; and Sieciechowicz et al., 1988, Phytochemistry
27:663-671. Because of its relative stability, asparagine does not
directly participate in nitrogen metabolism, but must be first
hydrolyzed by the enzyme asparaginase (ANS; E.C. 3.5.1.1) to
produce aspartate and ammonia which then could be utilized in
synthesis of amino acids and proteins (See FIG. 1).
[0013] 2.3. Plant Genes Involved in Nitrogen Assimilation and
Utilization
[0014] Many of the genes encoding enzymes involved in plant
nitrogen assimilation and utilization have been cloned and studied.
See Tsai and Coruzzi, Transgenic Plants for Studying Genes Encoding
Amino Acid Biosynthetic Enzymes, in Transgenic Plants, Vol. 1, Kung
and Wu eds., Academic Press, San Diego, Calif., (1993) pg 181-194,
and references cited therein for discussions of plant glutamine
synthetase (GS) and asparagine synthetase (AS) genes; Udvardi and
Kahn, 1991, Mol. Gen. Genet. 231:97-105, for a discussion of the
alfalfa aspartate aminotransferase gene; Zehnacker et al., 1992,
Planta 187:266-274, for a discussion of the tobacco glutamate
2:oxoglutarate aminotransferase (GOGAT, also known as glutamate
synthetase) gene; Lough et al, 1992, Plant Mol. Biol. 19:391-399,
and Dickson et al., 1992, Plant Mol. Biol. 20:333-336, for
discussions of lupin asparaginase gene.
[0015] Among the plant nitrogen assimilation and utilization genes,
the most extensively studied are the glutamine synthetase and
asparagine synthetase genes. Multiple genes exist for GS and AS,
and molecular characterization of these genes has shown that they
have different expression patterns.
[0016] 2.3.1. Glutamine Synthetase Genes
[0017] GS is active in a number of organs during plant development
(McNally et al., 1983, Plant Physiol. 72:22-25). In roots it
assimilates ammonia derived from soil water (Oaks and Hirel, 1985,
Ann. Rev. Plant Physiol. 36:345-365), and in root nodules of
legumes, GS assimilates ammonia fixed by rhizobia (Cullimore et al.
1983, Planta 157:245-253). In cotyledons GS reassimilates
nitrogenous reserves mobilized during germination (Lea and Joy,
1983, Amino acid interconversion in germinating seeds. In: Recent
Advances in Phythochemistry: Mobilization of Reserves in
Germination, ed. Nozolillo et al., Plenum Press, p. 77-109), and in
leaves chloroplastic GS2 assimilates ammonia released in
photorespiration_(Givan et al. 1988, TIBS 13:433-437). The various
roles of GS are undertaken by different GS isoforms which are
derived from different genes that are expressed differentially
(Gebhardt et al. 1986, EMBO J. 5:1429-1435; Tingey et al. 1987,
EMBO J. 6:1-9).
[0018] In pea, Phaseolus, and Arabidopsis, chloroplastic GS2 is
encoded by a single nuclear gene, whereas multiple genes for
cytosolic GS exist in each of these species (Bennett et al. 1989,
Plant Mol. Biol. 12:553-565; Tingey et al. 1988, J. Biol. Chem.
263:9651-9657; Peterman and Goodman, 1991, Mol. Gen. Genet.
230:145-154). The analysis of the expression of these GS genes in
vivo and in transgenic host plants has helped unravel the roles of
the various GS isoforms in plant nitrogen metabolism.
[0019] The GS gene family in pea comprises four distinct but
homologous nuclear genes. Three encode cytosolic GS isoforms, and
one encodes the chloroplastic GS2 isoform (Tingey et al., 1987,
EMBO J. 6:1-9; Tingey et al., 1988, J. Biol. Chem. 263:9651-9657).
Northern blot analysis has demonstrated that the gene for
chloroplastic GS2 is expressed in leaves in a light-dependent
fashion due in part to phytochrome and in part to photorespiratory
effects (Edwards and Coruzzi, 1989, Plant Cell 1:241-248). The
three genes for cytosolic GS (GS1, GS3A and GS3B) also appear to
serve distinct roles. In roots cytosolic GS1 is the predominant
isoform, although it is also expressed in nodules. Cytosolic GS3A
and GS3B are highly expressed in nodules and also in cotyledons of
germinating seeds (Tingey et al., 1987, EMBO J. 6:1-9; Walker and
Coruzzi, 1989, Plant Physiol. 91:702-708). While the GS3A and GS3B
genes are near identical in sequence, gene specific S1-nuclease
analysis has revealed that GS3A expression is consistently higher
than that of GS3B (Walker and Coruzzi, 1989, Plant Physiol.
91:702-708). Using promoter-GUS fusions and transgenic plant
analysis it has been shown that chloroplastic GS2 is expressed only
in photosynthetic cell-types and that cytosolic GS3A is expressed
exclusively in the phloem cells of the vasculature in most organs.
GS3A is also strongly expressed in root and nodule meristems
(Edwards et al., 1990, Proc. Natl. Acad. Sci. USA. 87:3459-3463;
Brears et al., 1991, The Plant Journal, vol. 1, pp. 235-244). From
the tightly controlled regulation at cell-type and organ level it
appears that the various genes for GS fulfill non-overlapping roles
in ammonia assimilation.
[0020] 2.3.2. Asparagine Synthetase Genes
[0021] Two AS genes have been cloned from pea (AS1 and AS2); both
are expressed at highest levels in root nodules and cotyledons. AS1
and AS2 are both expressed in roots. AS2 is expressed
constitutively in roots, while AS1 is expressed only in roots of
dark-grown plants (Tsai and Coruzzi, 1990, EMBO J 9:323-332).
Furthermore, AS1 and AS2 are expressed in mature leaves of
dark-adapted plants, whereas their expression is inhibited by
light. This high level of AS gene expression in the dark
corresponds to the use of asparagine as a long-distance nitrogen
transport compound synthesized under conditions of reduced
availability of photosynthetic carbon (asparagine has a higher N:C
ratio than glutamine). Studies of AS1 promoter-GUS fusions in
transgenic plants have shown that the AS1 gene, like the GS3A gene,
is also expressed exclusively in phloem cells. From the tightly
controlled regulation at cell-type and organ level, it seems that
the various AS genes may also fulfill non-overlapping roles in
plant nitrogen metabolism.
[0022] 2.4. Genetic Engineering of Nitrogen Assimilation and
Utilization Processes in Plants
[0023] In plants, genetic engineering of nitrogen assimilation
processes has yielded varied results. In one case, expressing a
prokaryotic ammonium dependent asparagine synthetase (ASN-A) gene
in tobacco conferred resistance to various glutamine synthetase
(GS) inhibitors (Dudits et al., Transgenic plants expressing a
prokaryotic ammonium dependent asparagine synthetase, WO 9111524,
Aug. 8, 1991). These same plants also exhibited a number of growth
alterations including increased growth rate, accelerated plant
development, early flower development and increased green mass and
plant dry weight. The growth effect of ASN-A expression is
paradoxical as GS inhibitor treatments enhanced rather than
attenuated growth in the engineered plants.
[0024] By contrast, numerous studies examining overexpression of
glutamine synthetase (GS) have failed to report any positive effect
of the overexpression on plant growth. See Lea and Forde, 1994,
Plant Molec. Biol. 17:541-558; Eckes et al., 1989, Molec. Gen.
Genet. 217:263-268 (transgenic tobacco plants overexpressing
alfalfa GS); Hemon et al., 1990, Plant Mol. Biol. 15:895-904
(transgenic tobacco plants overexpressing bean GS in the cytoplasm
or mitochondria); Hirel et al., 1992, Plant Mol. Biol. 20:207-218
(transgenic tobacco plants overexpressing soybean GS in tobacco
plants). One study has reported observing increases in total
soluble protein content in transgenic tobacco plants overexpressing
the alfalfa GS1 gene. However, since this same study also reported
similar increases in total soluble protein content in transgenic
tobacco plants expressing antisense RNA to the GS1 gene, the
relationship between GS1 expression and the increase in soluble
protein appears unclear (Temple et al., 1993, Mol. Gen. Genet.
236:315-325). One clearly established effect of GS overexpression
in plants is resistance to phosphinothricin, a GS inhibiting
herbicide (Eckes et al. ibid.; Donn et al., 1984, J. Molec. Appl.
Genet. 2:621-635 (a phosphinothricin-resistant alfalfa cell line
contained amplification of the GS gene)). There also has been a
claim that plants engineered with overexpression of an alfalfa GS
gene grow more rapidly than unengineered plants (Eckes et al.,
1988, Australian Patent Office Document No.: AU-A-17321/88). The
claimed faster growth, however, occurs only under low- but not
normal- or high-nitrogen growth conditions. Moreover, it is unclear
whether the faster growth produce mature plants with greater
biomass or reproductive yield. Compare id. with Eckes et al., 1989,
Molec. Gen. Genet. 217:263-268.
3. SUMMARY OF THE INVENTION
[0025] The present invention relates to the production of
transgenic plants with altered expression levels and/or
cell-specific patterns of expression of key enzymes involved in
nitrogen assimilation and utilization (The respective roles of
these enzymes are shown in FIG. 1) so that the resulting plants
have enhanced nitrogen assimilation and/or utilization capacities
as well as improved agronomic characteristics. The present
invention particularly relates to altering the expression of
glutamine synthetases, asparagine synthetases, glutamate
2:oxoglutarate aminotransferases (glutamate 2:oxoglutarate
aminotransferase is also known as glutamate synthetase), aspartate
aminotransferases, glutamate dehydrogenases and asparaginases (see
FIG. 1).
[0026] The invention has utility in improving important agronomic
characteristics of crop plants. One of the improvements would be
the ability of the engineered plants to be productively cultivated
with lower nitrogen fertilizer inputs and on nitrogen-poor soil.
Additional improvements include more vigorous (i.e., faster) growth
as well as greater vegetative and/or reproductive yield under
normal cultivation conditions (i.e., non-limiting nutrient
conditions). To achieve these same improvements, traditional crop
breeding methods would require screening large segregating
populations. The present invention circumvent the need for such
large scale screening by producing plants many of which, if not
most, would have the desired characteristics.
[0027] According to the present invention, achieving the desired
plant improvements may require, in some instances, the ectopic
overexpression of a single gene or multiple genes encoding nitrogen
assimilation or utilization enzyme(s). The modified expression may
involve engineering the plant with any or several of the following:
a) a transgene in which the coding sequence for the enzyme is
operably associated to a strong, constitutive promoter; b)
additional copies of the native gene encoding the desired enzyme;
c) regulatory gene(s) that activates the expression of the desired
gene(s) for nitrogen assimilation or utilization; d) a copy of the
native gene that has its regulatory region modified for enhanced
expression; and e) a transgene which expresses a mutated, altered
or chimeric version of a nitrogen assimilation or utilization
enzyme.
[0028] In other instances, achieving the desired plant improvements
may require altering the expression pattern of a nitrogen
assimilation or utilization enzyme. The altered expression pattern
may involve engineering the plant with any or many of the
following: a) a transgene in which the coding sequence for the
enzyme is operably associated to a promoter with the desired
expression pattern (such promoters may include those considered to
have tissue or developmental-specific expression patterns); b)
modified regulatory genes that activates the expression of the
enzyme-encoding gene in the preferred pattern; c) a native copy the
enzyme encoding gene that has its regulatory region modified to
express in the preferred pattern.
[0029] In yet other instances, achieving the desired plant
improvements may require suppressing the expression level and/or
pattern of a nitrogen assimilation or utilization enzyme. The
suppression of expression may involve engineering the plant with
genes encoding antisense RNAs, ribozymes, co-suppression
constructs, or "dominant negative" mutations (see Herskowitz, 1987,
Nature 329:219-222 for an explanation of the mechanism of gene
suppression by dominant negative mutations). Further, gene
suppression may also be achieved by engineering the plant with a
homologous recombination construct that replaces the native gene
with a copy of a defective gene or enzyme-encoding sequence that is
under the control of a promoter with the desired expression level
and/or pattern.
[0030] In still other instances, achieving the desired plant
improvements may require expressing altered or different forms of
the enzymes in the nitrogen assimilation or utilization pathways.
Such efforts may involve developing a plant-expressible gene
encoding a nitrogen assimilation or utilization enzyme with
catalytic properties different from those of the corresponding host
plant enzymes and engineering plants with that gene construct. Gene
sequences encoding such enzymes may be obtained from a variety of
sources, including, but not limited to bacteria, yeast, algae,
animals, and plants. In some cases, such coding sequences may be
directly used in the construction of plant-expressible gene fusions
by operably linking the sequence with a desired plant-active
promoter. In other cases, the utilization of such coding sequences
in gene fusions may require prior modification by in vitro
mutagenesis or de novo synthesis to enhance their translatability
in the host plant or to alter the catalytic properties of the
enzymes encoded thereon. Useful alterations may include, but are
not limited to, modifications of residues involved in substrate
binding and/or catalysis. Desired alterations may also include the
construction of hybrid enzymes. For instance, the different domains
of related enzymes from the same organism or different organisms
may be recombined to form enzymes with novel properties.
[0031] In all instances, a plant with the desired improvement can
be isolated by screening the engineered plants for altered
expression pattern or level of the nitrogen assimilation or
utilization enzyme, altered expression pattern or level of the
corresponding MRNA or protein, altered nitrogen assimilation or
utilization. capacities, increased growth rate, enhanced vegetative
yield, or improved reproductive yields (e.g., more or larger seeds
or fruits). The screening of the engineered plants may involve
enzymatic assays and immunoassays to measure enzyme/protein levels;
Northern analysis, RNase protection, primer extension, reverse
transcriptase/PCR, etc. to measure mRNA levels; measuring the amino
acid composition, free amino acid pool or total nitrogen content of
various plant tissues; measuring growth rates in terms of fresh
weight gains over time; or measuring plant yield in terms of total
dry weight and/or total seed weight.
[0032] The present invention is based, in part, on the surprising
finding that enhancing the expression of nitrogen assimilation or
utilization enzymes in plants resulted in enhanced growth
characteristics, or improved vegetative or reproductive yields. The
invention is illustrated herein by the way of working examples in
which tobacco plants were engineered with recombinant constructs
encoding a strong, constitutive ant promoter, the cauliflower
mosaic virus (CaMV) 35S promoter, operably linked with sequences
encoding a pea glutamine synthetase (GS) gene or a pea asparagine
synthetase (AS) gene. RNA and protein analyses showed that a
majority of the engineered plants exhibited ectopic, overexpression
of GS or AS. The GS or AS overexpressing lines have higher nitrogen
contents, more vigorous growth characteristics, increased
vegetative yields or better seed yields and quality than the
control, wild-type plant.
3.1. DEFINITIONS
[0033] The terms listed below, as used herein, will have the
meaning indicated.
[0034] 35S=cauliflower mosaic virus promoter for the 35S
transcript
[0035] AS=Asparagine synthetase
[0036] AspAT=aspartate aminotransferase (also known as
.DELTA.AT)
[0037] CaMV=Cauliflower Mosaic Virus
[0038] cDNA=complementary DNA
[0039] DNA=deoxyribonucleic acid
[0040] GDH=glutamate dehydrogenase
[0041] gene fusion=a gene construct comprising a promoter operably
linked to a heterologous gene, wherein said promoter controls the
transcription of the heterologous gene
[0042] GOGAT=glutamate 2:oxoglutarate aminotransferase (alternately
known as glutamate synthetase) Fd-GOGAT=Ferredoxin-dependent
glutamate synthase
[0043] NADH-GOGAT=NADH-dependent glutamate synthase
[0044] GS=glutamine synthetase
[0045] heterologous gene=In the context of gene constructs, a
heterologous gene means that the gene is linked to a promoter that
said gene is not naturally linked to. The heterologous gene may or
may not be from the organism contributing said promoter. The
heterologous gene may encode messenger RNA (mRNA), antisense RNA or
ribozymes.
[0046] nitrogen non-limiting growth condition=A nitrogen
non-limiting growth condition is one where the soil or medium
contains or receives sufficient amounts of nitrogen nutrients to
sustain healthy plant growth. Examples of nitrogen non-limiting
growth conditions are provided in section 5.2.3. Moreover, one
skilled in the art would recognize what constitutes such soils,
media and fertilizer inputs for most species and varieties of
important crop and ornamental plants (see section 5.3.).
[0047] PCR=polymerase chain reaction
[0048] Progenitor plant=untransformed, wild-type plant
[0049] RNA=ribonucleic acid
4. DESCRIPTION OF THE FIGURES
[0050] FIG. 1. Pathway of nitrogen assimilation/metabolism in
plants. The major route for nitrogen assimilation is via glutamine
synthetase (GS) and glutamate synthase (GOGAT). Glutamate
dehydrogenase (GDH) is thought to function under conditions of
ammonia toxicity in the biosynthetic role, or may provide catalytic
amounts of glutamate to fuel the GS/GOGAT cycle. GDH probably is
more active in its catalytic role to release ammonia from glutamate
(e.g., during germination). Aspartate amino transferase (AspAT)
catalyzes a reversible reaction. Asparagine synthetase (AS) has two
activities; a glutamine-dependent activity and an ammonia-dependent
activity. Asparagine catabolism occurs via asparaginase (ANS) to
liberate aspartate and ammonia.
[0051] FIG. 2. Engineering a chimeric Fd/NADH GOGAT enzyme. Plant
ferredoxin-GOGAT (Fd-GOGAT) large subunit contains Fd-Binding
domain (diagonal cross-bars). Plant and E. coli NADH-GOGAT: large
subunit (open bar), small subunit contains NADH-binding domain
(vertical hatches). Chimeric Fd/NADH GOGAT is engineered to contain
the large subunit of Fd-GOGAT (Fd-binding domain) plus the small
subunit of the NADH-GOGAT of either plant or E. coli. The
engineering is done by making an in-frame translational fusion of a
sequence encoding a plant Fd-GOGAT and a sequence encoding a small
subunit of a plant or E. coli NADH-GOGAT, containing the
NADH-binding domain. The chimeric protein encodes a bispecific or
bifunctional GOGAT enzyme which can utilize either Fd or NADH as
the reductant.
[0052] FIG. 3. Maps of Binary Plant Expression Vectors. The binary
expression vectors pTEV4, pTEV5, pTEV8 and pTEV9 are derivatives of
pBIN19 (Bevan, 1984, Nucleic Acids Res. 12:8711-8721) constructed
for the high level expression of cDNAs in transgenic tobacco. For
details of construction see Section 6.1.1.
[0053] FIG. 4. Chimeric 35S CaMV-GS cDNA Constructs Transferred to
Transgenic Tobacco. Pea GS cDNAs were cloned into pTEV expression
vectors (see FIG. 3, and Section 6.1.1) for expression behind the
Strasbourg strain CAMV 35S promoter (35S). For GS3A and GS2,
"modified" clones were constructed incorporating introns from the
genomic sequence into the cDNAs (see Section--6.1.2.). Sources of
the GS cDNA clones were: GS2 (also known as (aka) GS185); GS1 (aka
GS299); GS3A (aka GS341) (Tingey et al., 1988, J. Biol. Chem.
263:9651-9657; Tingey et al., 1987, EMBO J. 6:1-9).
[0054] FIG. 5. Analysis of GS Protein in Primary (Ti) Transformants
Containing GS Transgenes. Top panel: Western analysis of GS
polypeptides in primary transformants. Lanes 1 and 2: primary
transformants Z17-6 and Z17-12 carrying the cytosolic GS3A gene
show overexpression and co-suppression phenotypes respectively.
Lanes 3-6: primary transformants Z41-20, Z54-2, Z54-7, and Z54-8
carrying the chloroplastic GS2 gene are all co-suppressed for
chloroplast GS2 (cf. GS). Controls are: TL--tobacco leaf, PL--pea
leaf, and PR-pea root. Total GS activities are shown (as
percentages relative to controls=(100%)) below the Western panel.
Bottom panel: Coomassie staining of RUBISCO large subunit protein
demonstrating approximately equal loading of samples.
ctGS-chloroplastic GS2 (-45 kD); cyGS-cytosolic GS (-38 kD).
[0055] FIG. 6. Analysis of GS Protein, RNA and Holoenzyme from T2
Progeny Transgenic Plants Containing Pea GS Transgenes. Of the four
T2 plants from each primary transformant typically analyzed, a
single representative plant was included in this figure. In the
case of Z17-9, the T2 progenies showed two different profiles and
both are shown (Z17-9A and Z17-9B). Controls: TL/T--tobacco leaf,
P--pea leaf. Panel A (upper): Western analysis of GS polypeptides
in transgenic plants. Panel A (lower): Coomassie staining of
RUBISCO large subunit protein to show approximately equal loading
of samples. Panel B (upper): Northern blots hybridized with the
approximate cDNA probes for GS1 (left), GS3A (center), and GS2
(right). Panel B (lower): Control hybridization with the pea rRNA
gene probe. Panel C: Non-denaturing gel and GS activity analysis
showing GS holoenzymes A*, B, and C in transgenic plants. GS
activities are expressed as percentages compared to controls
(control=100% activity).
[0056] FIG. 7A. Activity Gel Analysis of GS Holoenzymes. Protein
extracts from pea chloroplast (PC), pea root (PR), tobacco
chloroplast (TC) and tobacco roots (TR) demonstrating the migration
of chloroplastic- and cytosolic-enriched GS protein samples
relative to the migration of the holoenzymes of GS1 and GS3A
overexpressing plants. Lane 1: pea chloroplast protein (PC) has GS
holoenzyme B only; lane 2: pea root protein (PR) has GS holoenzyme
C only; lane 3: tobacco chloroplast protein (TC) has GS holoenzyme
B only; lane 4: tobacco root protein has GS holoenzyme C only. Lane
5: protein from plant Z17-7 (carrying the 35S-GS3A construction)
has GS holoenzymes A* and B; lane 5: protein from plant Z3-l
(carrying the 35S-GS1 construction) has GS holoenzymes B and C.
[0057] FIG. 7B. Western Analysis of GS Proteins Isolated from GS
Holoenzymes A*, B, and C. Holoenzymes A* and C observed in
transgenic tobacco overexpressing GS3A and GS1 were excised from
non-denaturing gels, re-extracted in protein isolation buffer, and
electrophoresed under denaturing conditions for Western analysis
using GS antibodies. Lane 1: tobacco leaf protein as control; lane
2:.GS holoenzyme A* from Z17-7; lane 3: isolated chloroplast GS2
(holoenzyme B) as control; lane 4: GS holoenzyme C from Z3-1.
[0058] FIG. 8. Western and Northern Analysis of GS Protein and RNA
in Transgenic Plants Selected for Growth Analysis Ectopically
Expressing either Cytosolic GS1 or GS3A. Upper panel: Western blot
for GS proteins. Lower panel: Northern blot for GS MRNA. Pl and Tl
are pea and tobacco leaf controls. Lanes 1 and 2, and 5 and 6 are
plants overexpressing GS1, and lanes 3 and 4, and 7 and 8 are
plants overexpressing GS3A. Transgenic plants to the left of the
broken line were analyzed in growth experiment A, and those to the
right were analyzed in growth experiment B. Corresponding probes
were used in the Northern blot; the left pea control was hybridized
to GS1, and the right-hand pea control was hybridized to GS3A.
[0059] FIG. 9. Increase in fresh weight of transgenic lines
overexpressing cytosolic GS1 (Z3) or cytosolic GS3A (Z17). Panel A:
The results of experiment A with transgenic lines Z3-1, Z3-2,
Z17-6, Z17-7, and a non-transformed control (C). Panel B: The
results of experiment B with transgenic lines Z3-3, Z3-4, Z17-3,
Z17-11, and two non-transformed controls (C1 and C2). This is a
graphic representation of data shown in Table 2, and analyzed
statistically in Table 3.
[0060] FIG. 10. Qualitative growth pattern of plants with altered
GS expression patterns. Plants in each panel were sown at the same
time and grown in soil for approximately three weeks. Control
panel: SR1 untransformed tobacco (100% GS activity). Z3-A1 panel:
Transgenic plants with overexpress GS1 (123% GS activity). Z17-B7
panel: Transgenic plant which overexpresses GS3 (107% GS activity).
Z54-A2 Panel: Transgenic plant co-suppressed for GS2 (28% GS
activity).
[0061] FIGS. 11A and 11B. Linear relationship between GS activity
and plant fresh weight or total leaf protein. T2 progenies of
primary transformants which showed no segregation of the Kan.sup.R
phenotype associated with the transgene were selected for growth
analysis. Kan.sup.R T2 plants were selected on MSK media (R. B.
Horsch, et al., Science 227:1229 (1985)) and transferred to sand at
18 days. Plants were subirrigated and surface fed every two days
with 50 mls of 1.times. Hoagland's solution (D. R. Hoagland et al.,
Circ. Calif. Agric. Exp. Stn. 347:461 (1938)) containing 10 mM
KNO.sub.3. For each line, eight T2 progenies were analyzed
individually for total plant fresh weight (grams), specific
activity of total leaf GS as determined by the transferase assay
(B. M. Shapiro, et al., Methods Enzymol. 17A:910 (1970)) and
protein/gram fresh weight. Plants analyzed were: Control, SR1
untransformed tobacco; Z54-4 co-suppressed by GS2; Z17-7
overexpressing GS3A; Z3-1 overexpressing GS1. FIG. 11A; Plant fresh
weight vs. GS activity. FIG. 11B; protein/gm fresh weight vs. GS
activity
[0062] FIG. 12. Chimeric 35 S CaMV-AS Constructs Transferred to
Transgenic tobacco. cDNAs for the AS1 gene and the gln.DELTA.AS1
gene were fused to the 35S promoter and nopaline synthase
transcriptional terminator for transfer to tobacco using the binary
expression vector pTEV5.
[0063] FIG. 13. Northern analysis of transgenic plants expressing
either AS1 or gln.DELTA.AS1. 10 .mu.g of total RNA isolated from
leaves of individual transformants was loaded in leach lane. Blots
were probed with the AS1 cDNA from pea. A positive control includes
AS mRNA in dark-grown pea leaves (PL). A negative control includes
AS mRNA in light-grown tobacco leaves (TL).
[0064] FIG. 14. Increase in fresh weight of transgenic lines
overexpressing AS1 and gln.DELTA.AS1 is expressed graphically from
week 3 to week 6 post-germination. This is a graphic representation
of data shown in Table (5) and analyzed statistically in Table
(6).
5. DETAILED DESCRIPTION OF THE INVENTION
[0065] The present invention relates to genetic engineering of
nitrogen metabolism in plants. In particular, the invention relates
to altering the enzymes involved in nitrogen assimilation or
utilization and/or their expression in order to engineer plants
with better growth characteristics, enriched nutritional qualities,
improved vegetative and yield and/or enhanced seed yield or
quality.
[0066] Accordingly--without intending to be limited to a particular
mechanism--the targets for engineering are genes encoding for
enzymes involved in the assimilation of ammonia into the amino
acids, glutamine, aspartate, asparagine or glutamate, or in the
utilization of these same amino acids in biosynthetic reactions.
The target genes include those encoding glutamine synthetase (GS),
asparagine synthetase (AS), glutamate 2:oxoglutarate
-aminotransferase (GOGAT), aspartate aminotransferase (AspAT),
glutamate dehydrogenase (GDH) and asparaginase -(ANS). See FIG. 1
for a diagram of the roles played by of these enzymes in nitrogen
assimilation and utilization.
[0067] These enzymes can be altered or their expression can be
enhanced, suppressed or otherwise modified (e.g., ectopic
expression) to engineer a plant with desirable properties. The
engineering is accomplished by transforming plants with nucleic
acid constructs described herein. The transformed plants or their
progenies are screened for plants that express the desired altered
enzyme or exhibit the desired altered expression of the nitrogen
assimilation or utilization enzyme, altered expression of the
corresponding MRNA, altered nitrogen assimilation or utilization
capacities, increased growth rate, enhanced vegetative yield,
and/or improved reproductive yields.
[0068] Engineered plants exhibiting the desired physiological
and/or agronomic changes can be used in plant breeding or directly
in agricultural production. These plants having one altered enzyme
also may be crossed with other altered plants engineered with
alterations in the other nitrogen assimilation or utilization
enzymes (e.g., cross a GS overexpressing plant to an AS
overexpressing plant) to produce lines with even further enhanced
physiological and/or agronomic properties compared to the
parents.
[0069] The invention is illustrated by working examples of plants
engineered for ectopic, overexpression of GS or AS. In all
instances, engineered plants that exhibit ectopic, overexpression
of GS or AS also show better growth characteristics, enriched
nutritional qualities, improved vegetative yield and/or enhanced
seed quality or yield over control, wild-type plants.
5.1. Alteration of Nitrogen Assimilatory and Utilization
Pathways
[0070] In accordance with one aspect of the present invention,
desirable plants may be obtained by engineering ectopic
overexpression of enzymes involved in initial assimilation of
ammonia into amino acids glutamine, asparagine or glutamate and
further conversion to aspartate. The term ectopic is used herein to
mean abnormal subcellular (e.g., switch between organellar and
cytosolic localization), cell-type, tissue-type and/or
developmental or temporal expression (e.g., light/dark) patterns
for the particular gene or enzyme in question. Such ectopic
expression does not necessarily exclude expression in tissues or
developmental stages normal for said enzyme but rather entails
expression in tissues or developmental stages not normal for the
said enzyme. The term overexpression is used herein to mean above
the normal expression level in the particular tissue, all and/or
developmental or temporal stage for said enzyme.
[0071] Key enzymes involved in assimilation of ammonia into
glutamine and its further metabolism into glutamate, aspartate, and
asparagine are: glutamine synthetase, asparagine synthetase,
glutamate 2:oxoglutarate aminotransferase, aspartate
aminotransferase, glutamate dehydrogenase and asparaginase. The
present invention provides that engineering ectopic overexpression
of one or more of these enzymes would produce plants with the
desired physiological and agronomic properties. In a preferred
embodiment, a plant is engineered for the ectopic overexpression of
glutamine synthetase or asparagine synthetase. For GS, where
cytosolic and chloroplastic forms of an enzyme exist, engineering
of enhanced expression of the cytosolic form is preferred. The
cytosolic form of GS includes both nodule-specific (e.g., pea GS3A
& B) and root-specific (e.g., pea GS1) enzymes. The engineering
of enhanced expression of "root-specific" cytosolic GS (e.g., pea
GS1) is especially preferred. The present invention also provides
for engineering that alters the subcellular localization of said
enzyme. For example, engineering a chloroplast target sequence onto
a cytosolic. enzyme such as AS, may improve nitrogen assimilation
in plants. This would be especially valuable in nesophyll cells to
reassimilate photorespiratory ammonia.
[0072] In accordance to another aspect of the present invention,
desirable plants may be obtained by engineering enhanced ammonia
incorporation though an alternate nitrogen assimilation pathway. In
particular, the engineering is accomplished by suppressing the
normal, major route of nitrogen assimilation through glutamine
synthetase. In plant species that encode multiple GS isozymes, this
may require the suppression of the endogenous GS genes. In
preferred embodiments, a plant engineered with suppressed GS
expression is further engineered for ectopic overexpression of an
alternative N-assimilatory enzyme such as asparagine synthetase
(AS) and/or glutamine dehydrogenase (GDH). In most preferred
embodiments, the GS and AS/GDH engineered plant is additionally
engineered for enhanced expression of one or more of the other
enzymes involved in nitrogen assimilation or utilization processes
(see FIG. 1).
[0073] In accordance with a third aspect of the present invention,
desirable plants may be obtained by engineering ectopic
overexpression of an enzyme involved in the utilization of
assimilated nitrogen. Embodiments of this aspect of the present
invention may involve engineering plants with ectopic
overexpression of enzymes catalyzing the use of glutamine,
glutamate and asparagine in catabolic reactions. In a preferred
embodiment, a plant is engineered for the ectopic overexpression of
asparaginase.
[0074] In accordance with a fourth aspect of the present invention,
desirable plants may be obtained by engineering the expression of
an altered, mutated, chimeric, or heterologous form of an enzyme
involved in the assimilation or utilization of nitrogen.
Embodiments of this aspect of the present invention may involve
engineering plants to express nitrogen assimilation or utilization
enzymes from a heterologous source (ie. an enzyme from a different
plant or organism, including animals and microbes). Additional
embodiments may involve developing nitrogen assimilation or
utilization enzymes that have increased efficiencies, for example,
in substrate binding, catalysis, and/or product release and
engineering plants to express such novel enzymes. These novel
enzymes may be developed by in vitro mutagenesis of key amino acid
residues affecting the aforementioned processes. Alternatively such
novel enzymes may be developed by recombining domains from related
enzymes. For example, a chimeric bifunctional GOGAT enzyme could be
engineered to contain both ferredoxin- and NADH-GOGAT activities by
splicing the NADH binding domain of NADH-GOGAT onto the Fd-GOGAT
gene (see FIG. 2). Such a chimeric GOGAT enzyme would have the
advantage of being able to utilize either NADH or ferredoxin as a
reductant in the GOGAT reaction. The ectopic expression of this new
enzyme may result in more efficient synthesis of glutamate. Another
example of enzyme modification presented herein (see Section 7.0)
is the engineering of an AS enzyme which has a domain deleted to
alter its substrate specificity.
[0075] In accordance to the present invention, controlling the
tissue and developmental expression patterns of the nitrogen
assimilation or utilization enzymes may be important to achieving
the desired plant improvements. In instances where plants are
engineered for ectopic overexpression of the enzymes involved in
the normal or alternative ammonia assimilation pathways, preferred
embodiments of the present invention involve effecting altered
expression in many or all parts of the plant. In instances where
plants are engineered for ectopic overexpression of enzymes
catalyzing the use of assimilated nitrogen, preferred embodiments
of the present invention limit such expressions to nitrogen "sink"
tissues and structures such as leaves and seeds.
5.2. Generating Transgenic Plants
5.2.1. Nucleic Acid Constructs
[0076] The properties of the nucleic acid sequences are varied as
are the genetic structures of various potential host plant cells.
The preferred embodiments of the present invention will describe a
number of features which an artisan may recognize as not being
absolutely essential, but clearly advantageous. These include
methods of isolation, synthesis or construction of gene constructs,
the manipulations of the gene constructs to be introduced into
plant cells, certain features of the gene constructs, and certain
features of the vectors associated with the gene constructs.
[0077] Further, the gene constructs of the present invention may be
encoded on DNA or RNA molecules. According to the present
invention, it is preferred that the desired, stable genotypic
change of the target plant be effected through genomic integration
of exogenously introduced nucleic acid construct(s), particularly
recombinant DNA constructs. Nonetheless, according to the present
inventions, such genotypic changes can also be effected by the
introduction of episomes (DNA or RNA) that can replicate
autonomously and that are somatically and germinally stable. Where
the introduced nucleic acid constructs comprise RNA, plant
transformation or gene expression from such constructs may proceed
through a DNA intermediate produced by reverse transcription.
[0078] The nucleic acid constructs described herein can be produced
using methods well known to those skilled in the art. Artisans can
refer to sources like Sambrook et al., 1989, Molecular Cloning: a
laboratory manual, Cold Spring Harbor Laboratory Press, Plainview,
N.Y. for teachings of recombinant DNA methods that can be used to
isolate, characterize, and manipulate the components of the
constructs as well as to built the constructs themselves. In some
instances, where the nucleic acid sequence of a desired component
is known, it may be advantageous to synthesize it rather than
isolating it from a biological source. In such instances, an
artisan can refer to teachings of the likes of Caruthers et al.,
1980, Nuc. Acids Res. Symp. Ser. 7:215-233, and of Chow and Kempe,
1981, Nuc. Acids Res. 9:2807-2817. In other instances, the desired
components may be advantageously produced by polymerase chain
reaction (PCR) amplification. For PCR teachings, an artisan can
refer to the like of Gelfand, 1989, PCR Technology, Principles and
Applications for DNA Amplification, H. A. Erlich, ed., Stockton
Press, N.Y., Current Protocols In Molecular Biology, Vol. 2, Ch.
15, Ausubel et al. eds., John Wiley & Sons, 1988.
5.2.1.1. Expression Constructs
[0079] In accordance to the present invention, a plant with ectopic
overexpression of a nitrogen assimilation or utilization enzyme may
be engineered by transforming a plant cell with a gene construct
comprising a plant promoter operably associated with a sequence
encoding the desired enzyme. (Operably associated is used herein to
mean that transcription controlled by the "associated" promoter
would produce a functional messenger RNA, whose translation would
produce the enzyme.) In a preferred embodiment of the present
invention, the associated promoter is a strong and non tissue- or
developmental-specific plant promoter (e.g. a promoter that
strongly expresses in many or all tissue types). Examples of such
strong, "constitutive" promoters include, but are not limited to,
the CaMV 35S promoter, the T-DNA mannopine synthetase promoter, and
their various derivatives.
[0080] In another embodiment of the present invention, it may be
advantageous to engineer a plant with a gene construct operably
associating a tissue- or developmental-specific promoter with a
sequence encoding the desired enzyme. For example, where expression
in photosynthetic tissues and organ are desired, promoters such as
those of the ribulose bisphosphate carboxylase (RUBISCO) genes or
chlorophyll a/b binding protein (CAB) genes may be used; where
expression in seed is desired, promoters such as those of the
various seed storage protein genes may be used; where expression in
nitrogen fixing nodules is desired, promoters such those of the
legehemoglobin or nodulin genes may be used; where root specific
expression is desired, promoters such as those encoding for
root-specific glutamine synthetase genes may be used (see Tingey et
al., 1987, EMBO J. 6:1-9; Edwards et al., 1990, Proc. Nat. Acad.
Sci. USA 87:3459-3463).
[0081] In an additional embodiment of the present invention, it may
be advantageous to transform a plant with a gene construct operably
associating an inducible promoter with a sequence encoding the
desired enzyme. Examples of such promoters are many and varied.
They include, but are not limited to, those of the heat shock
genes, the defense responsive gene (e.g., phenylalanine ammonia
lyase genes), wound induced genes (e.g., hydroxyproline rich cell
wall protein genes), chemically-inducible genes (e.g., nitrate
reductase genes, gluconase genes, chitinase genes, etc.),
dark-inducible genes (e.g., asparagine synthetase gene (Coruzzi and
Tsai, U.S. Pat. No. 5,256,558, Oct. 26, 1993, Gene Encoding Plant
Asparagine Synthetase) to name just a few.
[0082] In yet another embodiment of the present invention, it may
be advantageous to transform a plant with a gene construct operably
linking a modified or artificial promoter to a sequence encoding
the desired enzyme. Typically, such promoters, constructed by
recombining structural elements of different promoters, have unique
expression patterns and/or levels not found in natural promoters.
See e.g., Salina et al., 1992, Plant Cell 4:1485-1493, for examples
of artificial promoters constructed from combining cis-regulatory
elements with a promoter core.
[0083] In yet an additional embodiment of the present invention,
the ectopic overexpression of a nitrogen assimilation or
utilization enzyme may be engineered by increasing the copy number
of the gene encoding the desired enzyme. One approach to producing
a plant cell with increased copies of the desired gene is to
transform with nucleic acid constructs that contain multiple copies
of the gene. Alternatively, a gene encoding the desired enzyme can
be placed in a nucleic acid construct containing an
amplification-selectable marker (ASM) gene such as the glutamine
synthetase or dihydrofolate reductase gene. Cells transformed with
such constructs is subjected to culturing regimes that select cell
lines with increased copies of ASM gene. See Donn et al., 1984, J.
Mol. Appl. Genet. 2:549-562, for a selection protocol used to
isolate of a plant cell line containing amplified copies of the GS
gene. Because the desired gene is closely linked to the ASM gene,
cell lines that amplified the ASM gene would also likely to have
amplified the gene encoding the desired enzyme.
[0084] In one more embodiment of the present invention, the ectopic
overexpression of a nitrogen assimilation or utilization enzyme may
be engineered by transforming a plant cell with nucleic acid
construct encoding a regulatory gene that controls the expression
of the endogenous gene or an transgene encoding the desired enzyme,
wherein the introduced regulatory gene is modified to allow for
strong expression of the enzyme in the desired tissues and/or
developmental stages. synthetase promoter, and their various
derivatives.
5.2.1.2. Suppression Constructs
[0085] In accordance to the present invention, a desired plant may
be engineered by suppressing GS activity or the activities of other
enzymes in nitrogen assimilation/metabolism (FIG. 1). In an
embodiment, the suppression may be engineered by transforming a
plant cell with a gene construct encoding an antisense RNA
complementary to a segment or the whole of a host target RNA
transcript, including the mature target mRNA. In another
embodiment, target gene (e.g., GS mRNA) suppression may be
engineered by transforming a plant cell with a gene construct
encoding a ribozyme that cleaves a host target RNA transcript,
(e.g., GS RNA transcript, including the mature GS mRNA).
[0086] In yet another embodiment, target gene suppression may. be
engineered by transforming a plant cell with a gene construct
encoding the target enzyme containing a "dominant negative"
mutation. Preferred mutations are those affecting catalysis,
substrate binding (e.g., for GS, the binding site of glutamate or
ammonium ion), or product release. A useful mutation may be a
deletion or point-mutation of the critical residue(s) involved with
the above-mentioned processes. An artisan can refer to teachings
herein and of Herskowitz (Nature, 329:219-222, 1987) for approaches
and strategies to constructing dominant negative mutations.
[0087] For all of the aforementioned suppression constructs, it is
preferred that such gene constructs express with the same tissue
and developmental specificity as the target gene. Thus, it is
preferred that these suppression constructs be operatively
associated with the promoter of the target gene. Alternatively, it
may be preferred to have the suppression constructs expressed
constitutively. Thus, a strong, constitute promoter, such as the
CaMV 35S promoter, may also be used to express the suppression
constructs. A most preferred promoter for these suppression
constructs is a modified promoter of the target gene, wherein the
modification results in enhanced expression of the target gene
promoter without changes in the tissue or developmental
specificities.
[0088] In accordance with the present invention, desired plants
with suppressed target gene expression may also be engineered by
transforming a plant cell with a co-suppression construct. A
co-suppression construct comprises a functional promoter
operatively associated with a complete or partial coding sequence
of the target gene. It is preferred that the operatively associated
promoter be a strong, constitutive promoter, such as the CaMV 35S
promoter. Alternatively, the co-suppression construct promoter can
be one that expresses with the same tissue and developmental
specificity as the target gene. Such alternative promoters could
include the promoter of the target gene itself (e.g., a GS promoter
to drive the expression of a GS co-suppression construct).
[0089] According to the present invention, it is preferred that the
co-suppression construct encodes a incomplete target mRNA or
defective target enzyme, although a construct encoding a fully
functional target mRNA or enzyme may also be useful in effecting
co-suppression.
[0090] In embodiments, where suppression of most, if not all, GS
isozymes is desired, it is preferred that the co-suppression
construct encodes a complete or partial copy of chloroplastic GS
MRNA (e.g., pea GS2 mRNA). As disclosed herein (section 6.2.2.),
such constructs are particularly effective in suppressing the
expression of the target gene.
[0091] In accordance with the present invention, desired plants
with suppressed target gene expression may also be engineered by
transforming a plant cell with a construct that can effect
site-directed mutagenesis of the endogenous target gene. (See
Offringa et al., 1990, EMBO J. 9:3077-84; and Kanevskii et al.,
1990, Dokl. Akad. Nauk. SSSR 312:1505-1507) for discussions of
nucleic constructs for effecting site-directed mutagenesis of
target genes in plants.) It is preferred that such constructs
effect suppression of target gene by replacing the endogenous
target gene sequence through homologous recombination with none or
inactive coding sequence.
5.2.1.3. Other Features of Recombinant Nucleic Acid Constructs
[0092] The recombinant construct of the present invention may
include a selectable marker for propagation of the construct. For
example, a construct to be propagated in bacteria preferably
contains an antibiotic resistance gene, such as one that confers
resistance to kanamycin, tetracycline, streptomycin, or
chloramphenicol. Suitable vectors for propagating the construct
include plasmids, cosmids, bacteriophages or viruses, to name but a
few.
[0093] In addition, the recombinant constructs may include
plant-expressible selectable or screenable marker genes for
isolating, identifying or tracking of plant cells transformed by
these constructs. Selectable markers include, but are not limited
to, genes that confer antibiotic resistances (e.g., resistance to
kanamycin or hygromycin) or herbicide resistance (e.g., resistance
to sulfonylurea, phosphinothricin, or glyphosate). Screenable
markers include, but are not limited to, the genes encoding
.beta.-glucuronidase (Jefferson, 1987, Plant Molec Biol. Rep
5:387-405), luciferase (Ow et al., 1986, Science 234:856-859), B
and C1 gene products that regulate anthocyanin pigment production
(Goff et al., 1990, EMBO J 9:2517-2522).
[0094] In embodiments of the present invention which utilize the
Agrobacterium system for transforming plants (see infra), the
recombinant DNA constructs additionally comprise at least the right
T-DNA border sequence flanking the DNA sequences to be transformed
into plant cell. In preferred embodiments, the sequences to be
transferred in flanked by the right and left T-DNA border
sequences. The proper design and construction of such T-DNA based
transformation vectors are well known to those skilled in the
art.
5.2.2. Transformation of Plants and Plant Cells
[0095] According to the present invention, a desirable plant may be
obtained by transforming a plant cell with the nucleic acid
constructs described herein. In some instances, it may be desirable
to engineer a plant or plant cell with several different gene
constructs. Such engineering may be accomplished by transforming a
plant or plant cell with all of the desired gene constructs
simultaneously. Alternatively, the engineering may be carried out
sequentially. That is, transforming with one gene construct,
obtaining the desired transformant after selection and screening,
transforming the transformant with a second gene construct, and so
on. In preferred embodiments each gene constructs would be linked
to a different selectable or screenable marker gene so as to
facilitate the identification of plant transformants containing
multiple gene inserts. In another embodiment, several different
genes may be incorporated into one plant by crossing parental lines
engineered for each gene.
[0096] In an embodiment of the present invention, Agrobacterium is
employed to introduce the gene constructs into plants. Such
transformations preferably use binary Agrobacterium T-DNA vectors
(Bevan, 1984, Nuc. Acid Res. 12:8711-8721), and the co-cultivation
procedure (Horsch et al., 1985, Science 227:1229-1231). Generally,
the Agrobacterium transformation system is used to engineer
dicotyledonous plants (Bevan et al., 1982, Ann. Rev. Genet
16:357-384; Rogers et al., 1986, Methods Enzymol. 118:627-641). The
Agrobacterium transformation system may also be used to transform
as well as transfer DNA to monocotyledonous plants and plant cells.
(see Hernalsteen et al., 1984, EMBO J 3:3039-3041; Hooykass-Van
Slogteren et al., 1984, Nature 311:763-764; Grimsley et al., 1987,
Nature 325:1677-179; Boulton et al., 1989, Plant Mol. Biol.
12:31-40.; Gould et al., 1991, Plant Physiol. 95:426-434).
[0097] In other embodiments, various alternative methods for
introducing recombinant nucleic acid constructs into plants and
plant cells may also be utilized. These other methods are
particularly useful where the target is a monocotyledonous plant or
plant cell. Alternative gene transfer and transformation methods
include, but are not limited to, protoplast transformation through
calcium-, polyethylene glycol (PEG)- or electroporation-mediated
uptake of naked DNA (see Paszkowski et al., 1984, EMBO J
3:2717-2722, Potrykus et al. 1985, Molec. Gen. Genet. 199:169-177;
Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828;
Shimamoto, 1989, Nature 338:274-276) and electroporation of plant
tissues (D'Halluin et al., 1992, Plant Cell 4:1495-1505).
Additional methods for plant cell transformation include
microinjection, silicon carbide mediated DNA uptake (Kaeppler et
al., 1990, Plant Cell Reporter 9:415-418), and microprojectile
bombardment (see Klein et al., 1988, Proc. Nat. Acad. Sci. USA
85:4305-4309; Gordon-Kamm et al., 1990, Plant Cell 2:603-618).
[0098] According to the present invention, a wide variety of plants
and plant cell systems may be engineered for the desired
physiological and agronomic characteristics described herein using
the nucleic acid constructs of the instant invention and the
various transformation methods mentioned above. In preferred
embodiments, target plants and plant cells for engineering include,
but are not limited to, those of maize, wheat, rice, soybean,
tomato, tobacco, carrots, potato, sugar beets, sunflower, yam,
Arabidopsis, rape seed, and petunia.
5.2.3. Selection and Identification of Transformed Plants and Plant
Cells
[0099] According to the present invention, desired plants may be
obtained by engineering the disclosed gene constructs into a
variety of plant cell types, including but not limited to,
protoplasts, tissue culture cells, tissue and organ explants,
pollens, embryos as well as whole plants. In an embodiment of the
present invention, the engineered plant material is selected or
screened for transformants (those that have incorporated or
integrated the introduced gene construct(s)) following the
approaches and methods described below. An isolated transformant
may then be regenerated into a plant. Alternatively, the engineered
plant material may be regenerated into a plant or plantlet before
subjecting the derived plant or plantlet to selection or screening
for the marker gene traits. Procedures for regenerating plants from
plant cells, tissues or organs, either before or after selecting or
screening for marker gene(s), are well known to those skilled in
the art.
[0100] A transformed plant cell, callus, tissue or plant may be
identified and isolated by selecting or screening the engineered
plant material for traits encoded by the marker genes present on
the transforming DNA. For instance, selection may be performed by
growing the engineered plant material on media containing
inhibitory amount of the antibiotic or herbicide to which the
transforming gene construct confers resistance. Further,
transformed plants and plant cells may also be identified by
screening for the activities of any visible marker genes (e.g., the
.beta.-glucuronidase, luciferase, B or C1 genes) that may be
present on the recombinant nucleic acid constructs of the present
invention. Such selection and screening methodologies are well
known to those skilled in the art.
[0101] Physical and biochemical methods also may be also to
identify plant or plant cell transformants containing the gene
constructs of the present invention. These methods include but are
not limited to: 1) Southern analysis or PCR amplification for
detecting and determining the structure of the recombinant DNA
insert; 2) Northern blot, S1 RNase protection, primer-extension or
reverse transcriptase-PCR amplification for detecting and examining
RNA transcripts of the gene constructs; 3) enzymatic assays for
detecting enzyme or ribozyme activity, where such gene products are
encoded by the gene construct; 4) protein gel electrophoresis,
Western blot techniques, immunoprecipitation, or enzyme-linked
immunoassays, where the gene construct products are proteins.
Additional techniques, such as in situ hybridization, enzyme
staining, and immunostaining, also may be used to detect the
presence or expression of the recombinant construct in specific
plant organs and tissues. The methods for doing all these assays
are well known to those skilled in the art.
5.2.4. Screening of Transformed Plants for those with Improved
Agronomic Traits
[0102] According to the present invention, to obtain plants with
improved agronomic characteristics, the transformed plants may be
screened for those exhibiting the desired physiological alteration.
For example, where the plants have been engineered for ectopic
overexpression of a GS enzyme, transformed plants are examined for
those expressing the GS enzyme at the desired level and in the
desired tissues and developmental stages. Where the plants have
been engineered for suppression of a target gene, transformed
plants are examined for those expressing the target gene product
(e.g., RNA or protein) at reduced levels in various tissues. The
plants exhibiting the desired physiological changes, e.g., ectopic
GS overexpression or GS suppression, may then be subsequently
screened for those plants that have the desired agronomic
changes.
[0103] Alternatively, the transformed plants may be directly
screened for those exhibiting the desired agronomic changes. In one
embodiment, such screening may be for productive growth of the
transformed plants under nitrogen nutrient deficient conditions.
That is screen for growth of transformed plants under conditions,
with respect to the available nitrogen nutrient, that cause the
growth of wild-type plant to cease or to be so diminished as to
significantly reduce the size or quality of the wild-type plant. An
example of a nitrogen nutrient deficient condition for tobacco and
plants with similar nitrogen nutrient requirements is that where
the sole nitrogen nutrient in the soil or synthetic medium is (a)
nitrate supplied or periodically applied at a concentration of 0.5
mM or lower, or (b) physiological equivalents of nitrate (e.g.,
ammonium or a mix of nitrate and ammonium) supplied or periodically
applied at a concentration that is physiologically equivalent to
0.5 mM nitrate or lower (see Eckes et al., 1988, Australian Patent
Office document no. AU-A-17321/88). Another example of a nitrogen
nutrient deficient condition is that where the steady state level
of the available nitrogen nutrient in the soil or synthetic medium
is less than about 0.02 mM nitrate or physiological equivalents
thereof. The term nitrate as used herein means any one or any mix
of the nitrate salts commonly used as plant nitrogen fertilizer,
e.g., potassium nitrate, calcium nitrate, sodium nitrate, ammonium
nitrate, etc. The term ammonium as used herein means any one or any
mix of the ammonium salts commonly used as plant nitrogen
fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium
sulfate, etc.
[0104] In other embodiments, the screening of the transformed
plants may be for improved agronomic characteristics (e.g., faster
growth, greater vegetative or reproductive yields, or improved
protein contents, etc.), as compared to unengineered progenitor
plants, when cultivated under nitrogen non-limiting growth
conditions (i.e., cultivated using soils or media containing or
receiving sufficient amounts of nitrogen nutrients to sustain
healthy plant growth). An example of nitrogen non-limiting
conditions for tobacco and plants with similar nitrogen nutrient
requirements is that where the sole nitrogen nutrient in soil or
synthetic medium is (a) nitrate supplied or periodically applied at
a concentration of 10 mM or higher, or (b) physiological
equivalents of nitrate supplied or periodically applied at a
concentration that is physiologically equivalent to 10 mM nitrate
or higher. Another example of nitrogen non-limiting conditions is
that where the steady state level of the available nitrogen
nutrient in the soil or synthetic medium is at least about 1.0 mM
potassium nitrate or physiological equivalents thereof. Additional
guidance with respect to what are nitrogen nutrient deficient or
"non-limiting" conditions for plant growth may be found in the art.
See for example, Hewitt, E. J., Sand and Water Culture Methods Used
in the Study of Plant Nutrition, 2nd ed., Farnham Royal (Bucks),
Commonwealth Agricultural Bureaux, 1966; and Hewitt, E. J., Plant
Mineral Nutrition, London, English University Press, 1975.
[0105] In enbodiments where the transformed plants are legumes,
direct screenings for transformed plants with the desired agronomic
changes and improvements may be conducted as described above but
under conditions where nodule formation or nitrogen-fixation is
suppressed.
[0106] According to the present invention, plants engineered with
the alterations in nitrogen assimilation or utilization processes
may exhibit improved nitrogen contents, altered amino acid or
protein compositions, vigorous growth characteristics, increased
vegetative yields or better seed yields and qualities. Engineered
plants and plant lines possessing such improved agronomic
characteristics may be identified by examining any of following
parameters: 1) the rate of growth, measured in terms of rate of
increase in fresh or dry weight; 2) vegetative yield of the mature
plant, in terms of fresh or dry weight; 3) the seed or fruit yield;
4) the seed or fruit weight; 5) the total nitrogen content of the
plant; 6) the total nitrogen content of the fruit or seed; 7) the
free amino acid content of the plant; 8) the free amino acid
content of the fruit or seed; 9) the total protein content of the
plant; and 10) the total protein content of the fruit or seed. The
procedures and methods for examining these parameters are well
known to those skilled in the art.
[0107] According to the present invention, a desired plant is one
that exhibits improvement over the control plant (i.e., progenitor
plant) in one or more of the aforementioned parameters. In an
embodiment, a desired plant is one that shows at least 5% increase
over the control plant in at least one parameter. In a preferred
embodiment, a desired plant is one that shows at least 20% increase
over the control plant in at least one parameter. Most preferred is
a plant that shows at least 50% increase in at least one
parameter.
5.3. Utility of the Invention
[0108] The engineered plants of the present invention may be
productively cultivated under nitrogen nutrient deficient
conditions (i.e., nitrogen-poor soils and low nitrogen fertilizer
inputs) that would cause the growth of wild-type plants to cease or
to be so diminished as to make the wild-type plants practically
useless. The engineered plants also may be advantageously used to
achieve earlier maturing, faster growing, and/or higher yielding
crops and/or produce more nutritious foods and animal feedstocks
when cultivated using nitrogen non-limiting growth conditions
(i.e., soils or media containing or receiving sufficient amounts of
nitrogen nutrients to sustain healthy plant growth). Nitrogen
non-limiting growth conditions vary between species and for
varieties within a species. However, one skilled in the art knows
what constitute nitrogen non-limiting growth conditions for the
cultivation of most, if not all, important crop and ornamental
plants. For example, for the cultivation of wheat see Alcoz et al.,
Agronomy Journal 85:1198-1203 (1993), Rao and Dao, J. Am. Soc.
Agronomy 84:1028-1032 (1992), Howard and Lessman, Agronomy Journal
83:208-211 (1991); for the cultivation of corn see Tollenear et
al., Agronomy Journal 85:251-255 (1993), Straw et al., Tennessee
Farm and Home Science: Progress Report, 166:20-24 (Spring 1993),
Miles, S. R., J. Am. Soc. Agronomy 26:129-137 (1934), Dara et al.,
J. Am. Soc. Agronomy 84:1006-1010 (1992), Binford et al., Agronomy
Journal 84:53-59 (1992); for the cultivation of soybean see Chen,
et al., Canadian Journal of Plant Science 72:1049-1056 (1992),
Wallace et al. Journal of Plant Nutrition 13:1523-1537 (1990); for
the cultivation of rice see Oritani and Yoshida, Japanese Journal
of Crop Science 53:204-212 (1984); for the cultivation of linseed
see Diepenbrock and Porksen, Industrial Crops and Products
1:165-173 (1992); for the cultivation of tomato see Grubinger et
al., Journal of the American Society for Horticultural Science
118:212-216 (1993), Cerne, M., Acta Horticulture 277:179-182,
(1990); for the cultivation of pineapple see Magistad et al. J. Am.
Soc. Agronomy 24:610-622 (1932), Asoegwu, S. N., Fertilizer
Research 15:203-210 (1988), Asoegwu, S. N., Fruits 42:505-509
(1987), for the cultivation of lettuce see Richardson and
Hardgrave, Journal of the Science of Food and Agriculture
59:345-349 (1992); for the cultivation of mint see Munsi, P. S.,
Acta Horticulturae 306:436-443 (1992); for the cultivation of
camomile see Letchamo, W., Acta Horticulturae 306:375-384 (1992);
for the cultivation of tobacco see Sisson et al., Crop Science
31:1615-1620 (1991); for the cultivation of potato see Porter and
Sisson, American Potato Journal, 68:493-505 (1991); for the
cultivation of brassica crops see Rahn et al., Conference
"Proceedings, second congress of the European Society for Agronomy"
Warwick Univ., p.424-425 (Aug. 23-28, 1992); for the cultivation of
banana see Hegde and Srinivas, Tropical Agriculture 68:331-334
(1991), Langenegger and Smith, Fruits 43:639-643 (1988); for the
cultivation of strawberries see Human and Kotze, Communications in
Soil Science and Plant Analysis 21:771-782 (1990); for the
cultivation of songhum see Mahalle and Seth, Indian Journal of
Agricultural Sciences 59:395-397 (1989); for the cultivation of
plantain see Anjorin and Obigbesan, Conference "International
Cooperation for Effective Plantain and Banana Research" Proceedings
of the third meeting. Abidjan, Ivory Coast, p. 115-117 (May 27-31,
1985); for the cultivation of sugar cane see Yadav, R. L.,
Fertiliser News 31:17-22 (1986), Yadav and Sharma, Indian Journal
of Agricultural Sciences 53:38-43 (1983); for the cultivation of
sugar beet see Draycott et al., Conference "Symposium Nitrogen and
Sugar Beet" International Institute for Sugar Beet
Research--Brussels Belgium, p. 293-303 (1983). See also Goh and
Haynes, "Nitrogen and Agronomic Practice" in Mineral Nitrogen in
the Plant-Soil System, Academic Press, Inc., Orlando, Florida, p.
379-468 (1986), Engelstad, O. P., Fertilizer Technology and Use,
Third Edition, Soil Science Society of America, p.633 (1985), Yadav
and Sharmna, Indian Journal of Agricultural Sciences, 53:3-43
(1983).
[0109] GS suppression have utility in that some GS suppressed
plants, particularly legumes, may grow faster or have higher
nitrogen contents than non-suppressed plants. (See Knight and
Langston-Unkefer, Science 241:951-954). GS suppressed plants may
also have altered amino acid or protein contents, making such
plants useful in preparation of special dietary foods. Further, all
the engineered plants disclosed herein may also serve as breeding
stocks for developing agriculturally useful plant lines.
6. EXAMPLE
Ectopic Overexpression of Glutamine Synthetase in Plants Causes an
Increase in Plant Growth Phenotype
[0110] Described herein is a molecular-genetic approach to
manipulate nitrogen use efficiency in transgenic plants. The
approach relies on the ectopic expression of glutamine synthetase,
that express GS in cell-types and/or at levels which the GS
expression is not normally found. The pattern of cell-specific GS
expression in transgenic plants is altered by constitutively
overexpressing the cytosolic GS (which is normally only expressed
in phloem) in all cell-types. Such ectopic expression of GS may
circumvent physiological limitations which result from the
compartmentalization and cell-type specificity of nitrogen
assimilatory enzymes. The ectopic high-level expression of
cytosolic GS in mesophyll cells might provide an alternate route
for the reassimilation of ammonia lost via photorespiration. This
may provide a growth advantage as the amount of ammonia lost via
photorespiration exceeds primary nitrogen assimilation by 10-fold
(Wallsgrove et al., 1983, Plant Cell Environ. 6:301-309; Keys et
al., 1978, Nature, 275:741-743). The studies disclosed herein show
that constitutive overexpression of a heterologous GS subunit for
cytosolic GS leads to increases in GS mRNA, GS protein, total GS
activity, native GS holoenzyme, and, in one case, to the production
of a novel GS holoenzyme. Transformed plants which overexpress
cytosolic GS have a statistically significant growth advantage
compared to wild type. They grow faster, attain a higher final
fresh weight and have more soluble proteins than untransformed
progenitor plants during the vegetative stage of their development.
In some instances, however, overexpression of cytosolic GS and/or
chloroplastic GS leads to a down regulation of endogenous gene
expression or co-suppression. Some transformed plants containing
cytosolic GS overexpression constructs and all transformed plants
containing chloroplastic GS2 constructs do not overexpress GS, but
rather are suppressed for GS expression, including suppression of
the endogenous GS gene (i.e., co-suppression). Such GS
co-suppressed plants may show poorer growth characteristics, but
may have altered amino acid and protein contents due to shunting of
nitrogen into other nitrogen assimilation/metabolism pathways.
6.1. Material and Methods
6.1.1. Plant Expression Vector Construction
[0111] Plant expression vectors pTEV 4,5,7, and 8 were constructed
as follows. A HindIII-EcoRI fragment containing the 35S promoter
from the Strasbourg strain of the Cauliflower mosaic virus (CaMV)
extending from -941 to +26 relative to the start of transcription
was. inserted into pBluescript KS II-(pT109) (Hohn et al., 1982,
Curr. Topics Microbiol. Immunol. 96:194-236). The polylinker
sequence between the HindIII and XhoI sites was then modified to
include Xbal, SstI, and StuI sites (pT145). This enabled a T4
polymerase-treated SstI-EcoRI fragment derived from pBIN19
(Clontech) and containing the nopaline synthase transcriptional
terminator to be inserted at the StuI site creating pT161. The
expression cassette thus constructed was flanked by EcoRI sites and
was transferred to pW3, a plasmid derived from pBIN19 (Bevan, 1984,
Nucleic Acids Res. 12:8711-8721) containing a modified polylinker.
A clone oriented with the 5' end of the promoter adjacent to the
left border of pW3 was selected (pW63) and numerous cloning sites
were inserted between promoter and terminator. This created the
following binary vectors with the unique cloning sites listed (FIG.
3): pTEV4 (HindIII-XbaI-BamHI-XhoI), pTEV5
(HindIII-StuI-SstI-KpnI), pTEV8 (HindIII-XhoI-BamHI-Xbal), pTEV9
(HindIII-KpnI-SstI-StuI).
6.1.2. Transfer of GS cDNAs to Binary Expression Vectors
[0112] CDNAs corresponding to the pea genes for cytosolic GS1 and
GS3A, and chloroplastic GS2 were transferred from pBluescript to
the binary expression vectors described above (see FIG. 4). These
cDNAs have previously been described as GS299, GS341, and GS185
respectively (Tingey et al., 1987, EMBO J. 6:1-9; Tingey et al.,
1988, J. Biol. Chem. 263:9651:9657). For chloroplastic GS2, a
modified cDNA was constructed which incorporated the first intron
of the genomic sequence into the cDNA at the appropriate position-
(54). This was made using the polymerase chain reaction to amplify
a fragment extending from the 5' end of the CDNA to the BsmI site
located within exon 2 (at amino acid 43), which could then be
cloned into the CDNA in pBluescript. For cytosolic GS3A a modified
cDNA (Z17) was constructed by exchange-cloning a BgIII-KpnI
fragment from a genomic GS3A clone into the pBluescript cDNA clone
generating a cDNA sequence into which all genomic introns (from
amino acid 6 onwards) had been inserted. The purpose of
constructing cDNA incorporating introns was to attempt to enhance
expression in transgenic plants as has been shown in monocots
(Sinibaldi and Mettler, 1991). The cDNAs were transferred from
pBluescript to the following binary expression vectors: GS1- pTEV4
into XbaI-XhoI sites to four pZ3 (NRRL Accession No. B-21330); GS3A
and modified GS3A-pTEV4 into XbaI-XhoI sites to form, respectively,
pZ9 (NRRL Accession No. B-21331) and pZ17 (NRRL Accession No.
21332); GS2 and modified GS2- pTEV5 into StuI-KpnI sites to form
respectively, pZ41 (NRRL Accession No. B-21333) and pZ54 (NRRL
Accession No. B-31334).
6.1.3. Plant Transformations
[0113] Binary vector constructions were transferred into the
disarmed Agrobacterium strain LBA4404 by triparental mating using a
previously described procedure (Bevan, 1984, Nucleic Acids Res.
12:8711-8721). Nicotiana tabacum line SR1 was transformed by a leaf
inoculation procedure (Horsch et al., 1985, Science 227-1299-1231),
and regenerated shoots were selected on medium containing 200
.mu.g/ml kanamycin. Primary transformants were maintained in
sterile culture and subsequently grown to maturity in soil.
Transgenic seeds were sterilized in 10% sodium hypochlorite and
germinated on medium containing 100 .mu.g/ml kanamycin.
6.1.4. GS Protein and Enzyme Activity Analysis
[0114] Soluble proteins were extracted from tobacco and pea leaf
tissue as previously described (Tingey and Coruzzi, 1987, Plant
Physiol. 84:366-373). Proteins were denatured and separated in 12%
acrylamide by SDS-PAGE and electroblotted onto nitrocellulose.
Western analysis was undertaken using the ProtoBlot kit supplied by
Promega and a mixture of antibodies raised to tobacco chloroplast
GS2 and Phaseolus cytosolic GS (Hirel et al., 1984, Plant Physiol.
74:448-450; Lara et al., 1984, Plant Physiol. 76:1019-1023). Total
GS activity in transformants was determined using a previously
described ADP-dependent transferase assay (Shapiro and Stadtman,
1970, Methods Enzymol. 17A;910-922). Non-denaturing gel
electrophoresis followed a published protocol (Davis, 1964, Annals
New York Acad. Sci. 121:404-427) in conjunction with the
ADP-dependent transferase assay for GS isozyme detection.
6.1.5. RNA Analysis
[0115] RNA was isolated using "RNA matrix" from Bio101 following
the protocol suggested by the manufacturer. Total RNA was
electrophoresed in 40 mM triethanolamine, 2 mM EDTA and 3.2%
formaldehyde in 1.2% agarose (Thomas, 1983, Methods Enzymol.
100:255-266). Gels were soaked in 10 mM sodium phosphate and
capillary blotted onto Hybond-N nylon membrane (Amersham). cDNAs
were labelled either using the random primer plus extension reagent
labeling system supplied by NEN, and strand specific riboprobes
were made using the Stratagene RNA transcription kit. Aqueous
hybridizations were done according to the membrane manufacturer's
protocol, and blots were washed in 0.1.times. SSPE, 0.1%.times.
SDS.
6.1.6. Plant Growth Conditions
[0116] Progenies of primary transformants previously characterized
as expressing GS1 or GS3A at high levels were germinated on
Murashige-Skoog (MS) medium containing 100 .mu.g/ml kanamycin.
After 14 days kanamycin resistant seedlings were transferred to 4
inch pots filled with white sand, which were covered with Saran
Wrapt for approximately one week to prevent excessive transpiration
and enable seedlings to become established. Pots were irrigated
periodically with an excess of 1.times. Hoagland's solution
containing 10 mM potassium nitrate as the only nitrogen source.
Subsequently between three and seven plants were sacrificed for
fresh weight determination each week, continuing for a period of
four weeks until shading of neighbors was apparent. Plants were
grown under a light-dark cycle of 16-8 h with a temperature cycle
of 24-18.degree. C. Daytime light intensity was 1000 lux.
6.2. Results
6.2.1. GS Constructions Introduced into Transgenic Plants
[0117] Pisum sativum cDNAs for chloroplastic GS2 (aka GS185 (Tingey
et al., 1988, J. Biol. Chem. 263:9651-9657)), cytosolic GS1 (aka
GS299 (Tingey et al., 1988, J. Biol. Chem. 263:9651-9657)) and GS3A
(aka GS341 (Tingey et al., 1987, EMBO J. 6:1-9)) were inserted into
PTEV binary expression vectors (see FIGS. 3 and 4) for expression
behind the CaMV 35S promoter and transferred to transgenic tobacco.
For GS2 (construct Z54, FIG. 4) and GS3A (construct Z17, FIG. 4)
cDNAs incorporating one or more introns were constructed and
expressed behind the CaMV 35S promoter. The purpose of constructing
cDNAs incorporating introns was to attempt to enhance expression in
transgenic plants, as has been shown for monocots (Sinibaldi and
Mettler, 1991, Progress in Nucleic Acid Research and Molecular
Biology 42:1991). In addition, unmodified full-length GS cDNAs were
also expressed under the 35S-CaMV promoter for GS2 (Z41), G3A (Z9),
and GS1 (Z3) (see FIG. 4). For each of the 35S-CaMV-GS
constructions detailed in FIG. 4, at least eight primary (T1)
transformants were analyzed and representative samples are shown in
FIG. 5. For selected primary transformants, four
kanamycin-resistant T2 progeny plants were also analyzed (FIG. 6).
The analysis of T1 and T2 plants presented below includes Western
analysis (FIG. 5 and FIG. 6, panel A); Northern blot analysis (FIG.
6, panel B), GS holoenzyme analysis (FIG. 6, panel C), and GS
enzyme activity analysis (FIG. 6, panel C and Tables lA and lB) and
are representative of all the transgenic lines analyzed.
6.2.2. Analysis of Transgenic Plants Carrying 35S-chloroplastic GS2
Gene Fusions
[0118] Transgenic plants containing either of the 35S-GS2
constructs (Z41 or Z54; FIG. 4) were analyzed. Both the 35S-GS2
(Z41) and the modified (intron-containing) 35S-GS2 construct (Z54)
gave similar results for both primary T1 transformants and for T2
progeny plants. Western blot analysis of all primary transformants
revealed a significant reduction in the abundance of chloroplastic
GS2 polypeptide (ctGS) (FIG. 5 lanes 3-6), when compared to
wild-type tobacco (FIG.
1TABLE 1A Total GS Activity in Primary Transformants (T1) Z41:
35S-GS2 Z54: 35S-GS2 (modified) Z41-6 42 Z54-1 13 Z41-7 74 Z54-2 11
Z41-8 23 Z54-3 49 Z41-12 66 Z54-4 22 Z41-14 44 Z54-6 39 Z41-15 nd
Z54-7 25 Z41-16 65 Z54-8 23 Z41-18 29 Z54-9 25 Z41-20 35 Z54-10 33
Z41-23 76 Z41-24 32 Z41-25 67 Z41-27 29 Z41-32 22 Z41-33 85 Z17:
35S-GS3 (modified) Z3: 35S-GS1 Z17-3 138 Z3-1 nd Z17-6 127 Z3-2 nd
Z17-7 119 Z17-8 36 Z17-9 45 Z17-10 52 Z17-12 28 Z17-14 145 Total GS
activity was determined for primary transformants and are expressed
as percentages compared to SR1 wild-type (=100). nd - not
determined.
[0119]
2TABLE 1B Total GS Activity in Primary Transformants (T1) and their
Progenies (T2) T1 T2-mean T2-A -B -C -D Z41: 35S-GS2 Z41-15 nd 27
15 7 75 11 Z41-20 35 50 53 33 31 81 Z41-33 85 35 31 30 32 46 Z54:
35S-GS2 (modified) Z54-2 11 28 30 19 21 42 Z54-7 25 22 29 21 18 19
Z54-8 23 35 34 39 31 35 Z17: 35S-GS3A (modified) Z17-6 127 100 112
99 94 96 Z17-7 119 107 104 103 111 108 Z17-9 45 44 126 14 26 11
Z17-10 52 27 33 50 18 5 Z17-12 28 18 21 18 22 10 Z3: 35S-GS1 Z3-1
nd 123 108 129 113 140 Z3-2 nd 120 114 129 121 116 Total GS
activity was determined for primary transformants and four T2
progeny plants (labeled A-D). Activity is expressed in percentage
of SR1 wild-type (= 100). nd = not determined.
[0120] 5, lane TL). Since the polyclonal GS2 antibodies have been
shown to recognize both pea and tobacco GS2 (Tingey and Coruzzi,
1987, Plant Physiol. 84:366-373; Tingey et al., 1988, J. Biol.
Chem. 263:9651-9657) this reduction reflects a down-regulation of
both the host tobacco GS2 gene and also of the pea GS2 transgene.
No change in the abundance of the cytosolic GS polypeptides (cyGS)
was observed in these transformants (FIG. 5, lanes 3-6) compared to
control untransformed wild-type tobacco (FIG. 5, lane TL). For Z41,
all fourteen independent primary transformants were down-regulated
for total GS activity, with a high of 85% wild-type activity to a
low of 22% wild-type GS activity (Tables 1A and 1B). For the Z54
constructs, all nine independent primary transformants regenerated
were down-regulated to below 50% of wild-type GS activity, with a
range of 49% to 11% (Tables 1A and 1B). From these data, it is
apparent that the intron containing Z54 constructs were severely
co-suppressed. By contrast, the 241 construct was less efficient at
down-regulating endogenous tobacco chloroplastic GS2 and these
plants showed a wider range of co-suppression phenotypes (see
variation in GS activity amongst Z41 individuals in Tables 1A and
1B). Typically, plants co-suppressed for GS2 (Z54 or Z41) grew more
slowly than wild-type and developed intervenial chlorosis (see FIG.
10) due either to the toxicity associated with ammonia accumulation
during photorespiration, or glutamine deficiency. These
transformants were therefore similar to the previously described
GS2 mutants of barley (Wallsgrove et al., 1987, Plant Physiol.
83:155-158). Co-suppressed plants of either Z41 or Z54 type grown
in an atmosphere of elevated (1.2%) CO.sub.2 (to suppress
photorespiration), or supplemented with glutamine, showed less
severe symptoms, also supporting the conclusion that these plants
were deficient in GS2.
[0121] Four kanamycin-resistant T2 progeny plants from primary Z41
and Z54 transformants were also analyzed (FIG. 6). The results
obtained from Western analysis and for total GS activity for
progenies were similar to those observed for primary transformants
(FIG. 6, panel A, and Table 1B). FIG. 6 shows data for one
representative T2 progeny member for several Z54 or Z41 primary
transformants (FIG. 6, lanes 9-14). Western blot analysis of these
plants confirmed the low abundance of the chloroplast GS2 protein
(FIG. 6, panel A) and non-denaturing GS activity gel analyses
confirmed the reduced abundance of the GS2 holoenzyme (FIG. 6,
panel C, lanes 9-14) compared to wild-type tobacco (FIG. 6, panel
C, lane TL). Northern analysis showed that transcripts from the GS2
transgene were undetectable (FIG. 6, panel B, lanes 9-14) compared
to that present in control pea RNA (FIG. 6, panel B, lane P). These
results suggest the specific co-suppression of tobacco
chloroplastic GS2 from the insertion of a pea GS2 transgene. In
addition, the pea GS2 transgene was also silenced. Levels of
cytosolic GS mRNA and protein were unaffected in these GS2
co-suppressed plants.
6.2.3. Analysis of Transgenic Plants Carrying 35-S Cytosolic GS3A
Gene Fusions
[0122] Transgenic plants containing either type of 35S-GS3A
construct (Z17 or Z9; FIG. 4) were analyzed. For Z17 (the intron
containing line), of the thirteen independent primary transformants
analyzed for GS activity, six showed overexpression of GS activity
(119-145%) while seven showed co-suppression (52-28%) compared to
untransformed controls (100%) (Tables 1A and 1B). FIGS. 5 and 6
contain data for representative overexpressers and co-suppressed
lines of Z17. Transformant Z17-12 is co-suppressed for GS enzyme
activity (27% of wild-type) and both chloroplastic GS2 and
cytosolic GS proteins are low (FIG. 5, lane 2) compared to
wild-type tobacco (FIG. 5, lane TL). By contrast, transformant
Z17-6 has elevated levels of total GS activity (127%) and increased
levels of cytosolic GS protein (FIG. 5, lane 1) compared to
wild-type tobacco (FIG. 5, lane TL). Analysis of the T2 progeny of
other independent transformants revealed additional transformants
to be down-regulated for cytosolic GS protein (Z17-9B and Z17-10;
FIG. 6, Panel A, lanes 6 and 7), while others had elevated levels
of cytosolic GS (Z17-7 and Z17-9A; FIG. 6, Panel A, lanes 4 and 5).
The co-suppression phenomenon observed for the Z17 plants (Z17-9B,
Z17-10, and Z17-12) is clearly different to that observed for the
GS2 transformants (Z54 and Z41) in that both chloroplastic GS2 and
cytosolic GS are down-regulated in the GS3A co-suppressed plants
(cf. FIG. 6, panel A, lanes 6-8 with lanes 9-14). FIG. 6 shows that
co-suppression caused by 35S-GS3A (Z17-9B, Z17-10, Z17-12) is
accompanied by reduced GS abundance (from Western and GS activity
gel analysis; FIG. 6, panels A and C, lanes 6-8) and virtually
undetectable transcription of the GS3A transgene (from Northern
analysis; FIG. 6, panel B, lanes 6-8). In transformants
overexpressing the GS3A construct (Z17-6, Z17-7, and Z17-9A), the
GS3A transcript is very abundant (FIG. 6, panel B, lanes 3-5) and
this reflects the greater abundance of cytosolic GS detectable by
Western blot analysis (FIG. 6, panel A, lanes 3-5) and GS activity
assays (Table 1). Non-denaturing GS activity gel analysis of
soluble proteins from these Z17 transformants which overexpress
cytosolic GS3A indicates the existence of a novel GS holoenzyme
(band A*, FIG. 6, panel C, lanes 3-5) which migrates more slowly
than the predominant chloroplast GS2 holoenzyme in wild-type
tobacco leaves (band B, FIG. 6, panel C, lane T). It is interesting
that individual Z17 transformants carrying the same GS3A transgene
construction should give two distinct phenotypes, one of
co-suppression (FIG. 6, lanes 6-8) and one of overexpression (FIG.
6, lanes 3-5).
[0123] To enlarge the size of the population of transgenic plants
analyzed, a second round of transformations was performed and
yielded results similar to those described above. Of a total of
twenty-three independent primary Z17 transformants analyzed, five
were co-suppressed for GS and eight overexpressed GS. In addition,
primary transformants were analyzed which contained an unmodified
(intron-less) GS3A cDNA (Z9, FIG. 4); of the four Z9 primary
transformants analyzed, one was co-suppressed for GS and two
overexpressed cytosolic GS. This suggested no. qualitative
difference between the Z17 (intron containing 35S-GS3A) and Z9
(35S-GS3A cDNA) constructions. Particularly intriguing is the
observation that Z17-9A and Z17-9B (FIG. 6, lanes 5 and 6) should
have diverse phenotypes as these two T2 plants were derived by
self-pollination from a single primary transformant. The Z17-9
primary transformant had been analyzed for total GS activity and
found to have reduced activity and therefore to be co-suppressed
(see Table 1). Two other T2 progeny plants of Z17-9 were analyzed
(Z17-9C and Z17-9D) and these were both found to be co-suppressed
giving a ratio of 3:1 in favor of co-suppression in this
population.
6.2.4. Analysis of Transgenic Plants Carrying the 35S-cytosolic GS1
Gene Fusion
[0124] Transgenic plants containing the 35S-GS1 construct (Z3; see
FIG. 4) were also analyzed. Of the eight independent Z3 primary
transformants, five gave a clear phenotype of overexpression from
Western and Northern blot analysis, and none were co-suppressed.
The T2 progeny of two of these Z3 transformants are shown in FIG.
6. Both Z3-1 and Z3-2 show an increased abundance of cytosolic GS
protein (FIG. 6, panel A, lanes 1 and 2) and this is reflected by
the increased levels of GS mRNA (FIG. 6, panel B, lanes 1 and 2).
Non-denaturing activity gel analysis demonstrated a GS holoenzyme
(band C) (FIG. 6, panel C, lanes 1 and 2) which migrated faster
than the chloroplastic GS2 holoenzyme of tobacco leaves (FIG. 6,
panel C, lane T). This faster migrating GS holoenzyme (band C) in
the Z3 plants corresponds in size to native pea cytosolic GS.
6.2.5. Analysis of Native and Novel Cytosolic GS Holoenzymes in
Transgenic Plants
[0125] Ectopic expression of cytosolic GS3A (Z17) and GS1 (Z3) gave
additional, but different, GS holoenzyme activity bands (e.g.,
bands A* and C) compared to chloroplast GS2 (band B) seen in
wild-type tobacco leaves (FIG. 6, panel C). Electrophoresis of
extracts from these transgenic plants was repeated in
non-denaturing activity gels including for comparison, lanes of pea
root (PR) and tobacco root (TR) protein which are enriched for the
cytosolic GS holoenzyme (band C) FIG. 7A, lanes 2 and 4), and
extracts derived from purified pea chloroplasts (PC) and tobacco
chloroplasts (TC) which are enriched for chloroplastic GS3
holoenzyme (band B) (FIG. 7A, lanes 1 and 3). The additional GS1
holoenzyme activity (band C) seen in extracts of leaves from
transgenic tobacco Z3-1 (FIG. 7A, lane 6) co-migrates with the
native pea cytosolic GS band (band C, FIG. 7A, lanes 2 and 4). By
contrast, the novel GS3A activity (band A*) seen in leaves of the
Z17-7 transgenic plants (FIG. 7A, lane 5) co-migrates with neither
the cytosolic GS (band C) nor the chloroplastic GS2 band (band B)
and is larger in size and more acidic in charge. To determine the
subunit composition of the GS activity bands A*, B, and C, these
bands were excised from preparative gels, and the extracted
proteins were reloaded on a denaturing SDS gel followed by Western
blot analysis for GS subunits (FIG. 7B). This analysis revealed
that both GS activity band A* and band C are comprised exclusively
of cytosolic GS polypeptides (FIG. 7B, lanes 2 and 4). This finding
discounted the possibility that the larger GS3A activity band A*
was the result of the assembly of heterologous GS3A cytosolic
subunits with endogenous tobacco pre-chloroplastic GS2 subunits. It
is possible that GS activity band A* represents the association of
transgenic GS3A subunits with a chaperonin-type protein, but
attempts to dissociate such a complex with ATP were unsuccessful.
Consequently, the nature of the novel GS holoenzyme remains
unclear.
6.2.6. Selection of Transformants Ectopically Overexpressing
Cytosolic GS1 or GS3A for Growth Analysis
[0126] Two sets of plants which ectopically overexpress cytosolic
GS3A (Z17) or cytosolic GS1 (Z3) were selected for growth analysis.
From the first round of transformations (see Experiment A, infra)
plants Z3-1 and Z3-2 were selected as GS1 high level expressers
(FIG. 6, lanes 1 and 2; FIG. 8, lanes 1 and 2), and plants Z17-6
and Z17-7 were selected as GS3A high level expressers (FIG. 5, lane
1; FIG. 6, lanes 3 and 4; FIG. 8, lanes 3 and 4). Kanamycin
resistant T2 progenies of these transformants were selected for
growth analysis in experiment A described below. From the second
round of transformations, two more independently transformed
GS1-overexpressing plants (Z3-3 and Z3-4); (FIG. 8, lanes 5 and 6),
and two more independently transformed GS3A-overexpressing plants
(Zl7-3 and Z17-1l) (FIG. 8, lanes 7 and 8) were selected for
analysis. The kanamycin-resistant T2 progenies of these plants were
analyzed in the second growth experiment (Experiment B, infra).
6.2.7. Design of Plant Growth Experiments
[0127] Plant growth analysis was undertaken on the T2 progeny
plants analyzed for GS protein and RNA in FIG. 8. Individual T2
plants were grown in white sand and growth was assessed by fresh
weight determination of 4-7 plants per time point. Fresh weight
measurements were taken only during the vegetative stage of growth
when plants were growing rapidly and were exclusively dependent on
supplied nitrogen and were not remobilizing large internal sources
of nitrogen as might occur during bolting and flowering. Plants
were grown under conditions where nitrogen was non-limiting (i.e.,
regular fertilization with 10 mM nitrate) and which reduced the
photosynthetic interference of neighboring plants, and the growth
analysis was terminated when such interference became apparent. All
plants analyzed were of the same age, and analysis stated at
between 0.1 and 0.3 g fresh weight, and continued until the plants
were approximately six weeks old when the interference of
neighboring plants became apparent at the onset of bolting.
6.2.8. Plant Growth Experiment A
[0128] Table 2 shows the results of mean total fresh weight
determinations for lines Z3-1 and Z3-2 (overexpressing GS1) and for
Z17-6 and Z17-7 (overexpressing GS3A). These results are expressed
graphically in FIG. 9, panel A and analyzed statistically in Table
3. All four transgenic lines overexpressing pea cytosolic GS
outgrew the control by between 35% and 114%, and this was
statistically significant for three lines; Z3-2(P=0.08),
Z17-6(P=0.0015) and Z17-7(P=0.013) (Table 3).
6.2.9. Plant Growth Experiment B
[0129] The growth experiment was repeated with different transgenic
lines carrying the same GS1 (Z3) and GS3A (Z17) constructions to
confirm the results obtained above, including larger plant
populations for statistical analysis. Table 2 shows the mean data
for four time points for transgenic lines Z3-3, Z3-4, Z17-3, and
Z17-11, together with two control lines (C1, C2). All lines
3TABLE 2 Growth Increase of Transgenic Lines Overexpressing
Cytosolic GS1 (Z3) or Cytosolic GS3a (Z17) Experiment A.sup.1 C
Z3-1 Z3-2 Z17-6 Z17-7 Week 4 0.42 0.33 0.42 0.44 0.52 Week 5 1.40
1.88 2.36 2.73 1.91 Week 6 4.06 5.48 8.67 8.27 6.80 % Increase at
100 135 214 204 150 week 6 compared to control Experiment B.sup.1
C-1* C-2* Z3-3 Z3-4 Z17-3 Z17-11 Week 3 0.12 0.07 0.32 0.24 0.32
0.20 Week 4 0.60 0.41 1.11 0.77 1.08 1.00 Week 5 1.19 1.11 1.82
1.36 2.39 1.71 Week 6 6.49 5.83 9.37 6.04 9.34 9.06 % Increase at
100 90 144 93 144 140 week 6 com- pared to C-1 .sup.1Mean total
fresh weight (in grams) of transgenic lines and controls measured
over a period of three to four weeks immediately prior to the onset
of bolting. *C-1 is control 1 and C-2 is control 2.
[0130]
4TABLE 3 Growth Increase of Transgenic Lines Over- expressing
Cytosolic GS1 (Z3) or Cytosolic GS3A (Z17) with Comparison to
Controls By Unpaired T Test Experiment A C Z3-1 Z3-2 Z17-6 Z17-7
week 6 4.06 5.48 8.67 8.27 6.80 % Increase at 100 135 214 204 150
week 6 compared to control Number of Plants 3 6 5 7 6 (week 6)
Standard Error 0.51 0.75 1.62 0.53 0.53 Standard 0.88 1.85 3.62
1.41 1.28 Deviation "T" for unpaired 1.23 2.10 4.72 3.29 test to
control (7) (6) (8) (7) (df) Probability 0.26 0.08 0.0015 0.013
Significance NS (*) ** * Experiment B C-1 C-2 Z3-3 Z3-4 Z17-3
Z17-11 week 6 6.49 5.83 9.37 6.04 9.34 9.06 % Increase at 100 90
144 93 144 140 week 6 to C-1 Number of Plants 7 6 7 7 7 7 (week 6)
Standard 0.60 1.07 0.88 0.61 1.06 0.73 Error Standard 1.58 2.61
2.33 1.61 2.82 1.94 Deviation Z3-3 Z3-4 Z17-3 Z17-11 "T" for 2.70
(12) 0.53 (12) 2.34 (12) 2.72 (12) unpaired test to C-1 (df)
Probability 0.019 0.61 0.038 0.019 Significance * NS * * Mean total
fresh weight for transgenic lines (in grams) and controls at week
6. The statistical analysis was done for the final week's
measurement only, and in the case of experiment II control-1 (C-1)
was selected for the T-test. df - degrees of freedom; The
probability of the populations being related was deemed to be
highly significant (**) for P (0.001, significant (*) for P (0.05,
and marginally significant ((*)) for P (0.01. NS = not
significant.
[0131] except Z3-4 outgrew controls by between 40 and 44% and the
difference in fresh weights at six weeks was statistically
significant (Table 3). These results are also shown graphically in
FIG. 9, panel B. It is apparent that the second growth experiment
corroborated the results of the first, suggesting that ectopic
overexpression of wither pea cytosolic GS1 or GS3A enhanced growth
rate in tobacco; in all lines tested GS3A overexpression gave an
increase in growth rate which was statistically significant
increases in growth rate to the transgenic tobacco, compared to
non-transformed controls.
6.2.10. Qualitative Effect of GS Overexpression on Plant Growth
[0132] FIG. 10 demonstrates a qualitative comparison of the growth
phenotype of plants which overexpress GS (Z3-A1 and Z17-B7) to
those of control plants and plants co-suppressed for GS (Z54-A2).
The results demonstrate that even low level GS overexpression
results in readily discernible growth improvements (FIG. 10,
compare the growth of Z17-B7 and Z3-A1 with that of control
plants). Moreover, these results show that the growth improvements
are due to GS overexpression and not to the mere engineering of
plants with CaMV-35S GS constructs. For example, Z54-A1, which as
been engineered with CaMV 35S-GS2 and was co-suppressed for GS
expression, exhibited profoundly poor growth. Furthermore, these
results demonstrate that GS activity is a rate limiting step in
plant growth as inhibition of this enzyme causes severe phenotypic
effects on growth.
6.2.11. Correlation Between GS Activity and Final Fresh Weight and
Total Protein
[0133] Experiments were performed to determine whether changes in
GS activity associated with ectopic overexpression or
co-suppression of GS genes had an effect on "final" fresh weight at
the end of the vegetative growth phase. Growth analysis was
performed on T2 generation plants for a line co-suppressed by GS2
(Z54-4), a line overexpressing GS1 (Z3-1), a line overexpressing
GS3A (Z17-7), and an untransformed tobacco control (SR1). Plants
were grown in sand and irrigated periodically with Hoagland's
solution containing 10 mM KNO.sub.3. At designated time-points,
eight individual T2 plants from each line were weighed and leaf GS
activity was determined for each individual. Analysis of this data
reveals a linear relationship between "final" fresh weight and GS
specific activity for all individuals assayed at both 32 days and
43 days (FIG. 11A). For example, Z54-4 plants which are
co-suppressed for GS activity (27% of wild-type GS activity) weigh
one-half as much as controls, while plants which overexpress GS3A
(136% GS activity) or GS1 (284% GS activity) out-weigh controls by
1.5-times and 2-times, respectively. For these same individual T2
plants, a linear relationship also exists between total leaf
protein (.mu.g protein/gm fresh weight) and leaf GS activity.
Plants expressing the highest levels of GS activity (284%) had
1.5-fold higher levels of soluble protein/gram fresh weight
compared to controls (FIG. 11B). An unpaired T-test analysis of
this data revealed that the GS overexpressing lines (Z3-1,Z17-7)
had significantly greater GS activity, fresh weight, and leaf
soluble protein with a p value of <0.0001, with the exception of
fresh weight for Z17-7 whose p value was 0.0007. Similarly the line
co-suppressed by GS2 (Z54-4) had significantly less GS activity,
fresh weight, and leaf soluble protein than control SR1 with a p
value of <0.0001. The GS activity profiles of the GS
overexpressing T2 lines used in the growth study (Z3,Z17) parallel
the parental TO lines and the T1 progeny, except that the GS
activities were consistently higher in the T2 generation. This is
most likely due to the fact that some or all of the transgenes
became homozygous in the T2 generation, as there was no observed
segregation of the Kan phenotype associated with the GS transgene.
At the end of the growth experiment, the transgenic lines
overexpressing GS were visibly greener and dramatically larger than
controls.
6.3. Discussion
[0134] As genetic engineering begins to assume significance in crop
plant improvement it is becoming increasingly important to
understand the parameters critical in the overexpression of
selected genes. It is apparent that the overexpression of genes for
which there are host plant homologs may be more complex than the
overexpression of genes for which there are no homologs, such as
viral coat protein and BT toxin genes (Powell-Abel et al., 1986,
Science 232:738-743; Vaeck et al., 1987, Nature 328:33-37). This is
due to the phenomenon of co-suppression in which the transgenic
plant can detect and silence a transgene to which there is a host
homolog, perhaps by feedback inhibition or some other mechanism
(van der Krol et al., 1990, Plant Cell 2:291-299; Napoli et al.,
1990, Plant Cell 2:279-289). Presented here is an effort to
ectopically overexpress three different pea GS genes for
chloroplast or cytosolic GS behind the same constitutive promoter
(35S-CaMV) in transgenic tobacco. The effort resulted in
overexpression and/or co-suppression that is different for each GS
gene. Furthermore, for the two genes for cytosolic GS which were
successfully overexpressed (GS1 and GS3A), the overexpression
resulted not only in over production of GS RNA, protein and enzyme,
but also in a phenotype of improved nitrogen use efficiency.
[0135] Overexpression of the pea gene for cytosolic GS1 in tobacco
gives a clear phenotype of increased GS activity, increased
cytosolic GS protein, and high levels of transgene mRNA.
Furthermore, the GS1 protein assembles into a GS holoenzyme similar
in size and charge to native pea cytosolic GS. In transgenic plants
overexpressing cytosolic GS3A, the situation is somewhat different.
High levels of GS3A transgene mRNA give rise to increased levels of
cytosolic GS which are visible on Western blots. However, the
overexpression of GS3A causes the appearance of a novel GS
holoenzyme which is larger than the native chloroplastic or
cytosolic GS holoenzymes of either pea or tobacco. In these
transgenic plants, the cytosolic GS gene was being expressed in a
cell type where it is not normally found (e.g., mesophyll cells),
and it was possible that the larger GS holoenzyme in the GS3A
transgenic leaves was due to the co-assembly of cytosolic GS
subunits with native pre-chloroplast GS2. However, this novel GS3A
holoenzyme was shown to be composed exclusively of cytosolic GS
subunits and was therefore not due to the co-assembly of transgenic
GS3A subunits with endogenous tobacco pre-chloroplastic GS2. Two
other possibilities exist. The larger GS3A holoenzyme may be the
result of transgenic GS3A subunits assembling into a configuration
other than their usual octameric structure. Alternatively, the
novel GS3A holoenzyme may result from the failure of the
overexpressed cytosolic subunits to be released from an assembling
chaperonin. Indeed, the close association of GS with groEL-like
proteins has previously been observed in pea (Tsuprun et al., 1992,
Biochim. Biophys. Acta 1099:67-73). However, our attempts to
dissociate the novel GS3A activity band from a potential chaperonin
using ATP were unsuccessful. Although the novel GS3A holoenzyme
must clearly possess GS activity (from its detection in GS activity
gel analysis) it is interesting to speculate whether or not this
novel GS isozyme possesses a similar activity to the native
cytosolic GS or chloroplastic GS2 holoenzymes on an equimolar
basis. If this is the case, it might be predicted that plants
overexpressing 35S-GS3A, and therefore possessing the novel GS
holoenzyme, may have elevated total GS activities. In fact this was
not the case; the mean total GS activity (compared to wild-type) of
four Z17-6 T2 progeny plants (expressing GS3A) was found to be
100%, and that of four Z17-7 progeny plants was 107% compared to
wild-type. By contrast, GS activity values obtained for T2
progenies of Z3-1 and Z3-2 (overexpressing a GS1 native holoenzyme)
were 123% and 120% respectively, compared to wild-type. This
suggests that the assembly in the GS1 subunits in the Z3
overexpressing transformants into a GS holoenzyme of native size
was advantageous to total GS activity.
[0136] Here, nitrogen use efficiency was assessed during the
vegetative growth stage of transgenic tobacco which successfully
overexpressed wither cytosolic GS1 or cytosolic GS3A. During
vegetative growth there is rapid leaf development characterized by
rapid nutrient uptake and the maximization of photosynthetic
capacity. Nitrogen is the most frequently limiting micronutrient,
and the physiology of its uptake and use within the plant differs
between the vegetative and generative stages. firstly there is
nitrogen incorporation from the soil, its incorporation into
expanding tissues, and the limitation of losses through
photorespiration and subsequently, with the onset of bolting, there
is the mobilization of nitrogen reserves for conversion to seed
yield during the generative stage of growth. It is likely that the
parameters of nitrogen use efficiency are less complex during the
vegetative growth stage of development, and our transgenic plant
growth analysis has focused on this stage of growth.
[0137] The present findings indicate that ectopically expressed pea
cytosolic glutamine synthetase in tobacco provides a considerable
advantage in the vegetative growth stage of transgenic tobacco.
Plants which overexpress either cytosolic GS1 or GS3A ectopically
(i.e., in all cell types) yield a higher total fresh weight that
controls. It was particularly striking that all GS3A expressing
lines (Z17) had higher total fresh weights than controls at six
weeks and these were always statistically significant. In each case
there was a less than a 5% chance that the difference between
control and transgenic lines was due to sample variance. For the
GS1 expressing lines analyzed (Z3), three out of four outgrew
controls and for two of these the difference was statistically
significant at the 10% level. This increased use efficiency of
nitrogen may enable crops to be similarly engineered to allow
better growth on normal amounts of nitrogen or cultivation with
lower fertilizer input, or alternatively on soils of poorer quality
and would therefore have significant economic impact in both
developed and developing agricultural systems.
[0138] Although GS-overexpression has previously been attempted in
transgenic tobacco (Eckes et al., 1989, Mol. Gen. Genet.
217:263-268; Hemon et al., 1990, Plant Mol. Biol. 15:895-904; Hirel
et al., 1992, Plant Mol. Biol. 20:207-218; Temple et al., 1993,
Mol. Gen. Genet. 236:315-325), this is the first report in which
overexpression of GS is correlated with a significant increase in
GS activity and an improvement in plant growth and nutritional
characteristics. Temple et al. reported increases in GS mRNA and
protein, but no corresponding increase in GS activity in the
transgenic plants (Temple et al., ibid). Hemon et al. reported
increased levels of GS mRNA in transgenic plants engineered with GS
expression constructs, but found no corresponding increase in GS
protein or enzyme activity (Hemon et al., ibid). In two other
reports, overexpression of GS genes in transgenic plants did result
in increased levels of GS enzyme, but the studies reported no
phenotypic effects of GS overproduction (Eckes et al., ibid; Hirel
et al., ibid). There is one report of overexpression of an alfalfa
GS gene improving plant growth rate by about 20% (Eckes et al.,
1988, Australia Patent Application No. AU-A-17321/88). However,
this reported improvement appears to be limited to growth under
low-nitrogen conditions only. Identically engineered plants were
reported to show no phenotypic changes, as compared to control
plants, in a subsequent analysis carried out on a nitrogen
non-limiting medium (Eckes et al., 1989, Mol. Gen. Genet.
217:263-268). In addition, there is no report that the faster rate
of growth results in difference in final fresh weight between the
engineered and control plants. In contrast to these earlier
studies, the instant invention demonstrate unequivovally that,
regardless of the nitrogen conditions, GS overexpression can
improve growth, yield, and/or nutritional characterisitics of
plants.
[0139] The agricultural utility of the instant invention is
directly relevant to crop species in which the vegetative organs
are harvested, and these include all forage crops, potato, sugar
beet, and sugar cane as well as tobacco. Within a week of the final
fresh weight recordings presented here, plants started to undergo
internode extension, and the standard deviation of subsequent fresh
weight measurements for each population increased as a result of
the differing physiological stage of plants. Whether the increased
vegetative growth rate would also lead to a significant seed yield
advantage is an important question which remains to be answered.
The physiological parameters relevant to seed yield and seed
nitrogen content include not only the efficiency of nitrogen
uptake, but also the remobilization of reserves at the onset of
bolting, and the consequences of field population density. Such
studies would be better undertaken in a transgenic species which
has been selected for seed yield and for which there is some
understanding of yield physiology.
[0140] The finding that co-suppression of endogenous tobacco GS by
genes encoding chloroplastic GS2 and cytosolic GS3A of pea, but not
by cytosolic GS1 is also intriguing. This is especially so as pea
GS2 suppresses only the tobacco chloroplastic GS2 form while GS3A
suppresses both tobacco chloroplastic GS2 and cytosolic GS.
Previously, Petunia chalcone synthase and
dihydroflavanol-4-reductase have been shown to co-suppress both
endogenous and transgenes in transgenic Petunia (van der Krol et
al., 1990, Plant Cell 2:291-299; Napoli et al., 1990, Plant Cell
2:279-289). More recently it has been reported that either the 5'
or the 3' end of the chalcone synthase gene was sufficient to cause
co-suppression, but that a promoter-less gene was not (Jorgensen,
1992, Agbiotech News and Information Sept:1992), suggesting the
necessity of transcriptional initiation. Transient ectopic sequence
pairing has been invoked as a possible mechanism for gene silencing
and this may depend on the unwinding of DNA presumably associated
with the initiation of transcription (Jorgensen, 1990, Trends in
Biotechnology 8:340-344; Jorgensen, 1991, Trends in Biotechnology
9:255-267; Jorgensen, 1992, Agbiotech News and Information
September:1992). From the present findings on pea GS gene
expression it appears that the co-suppression phenomenon does not
depend on perfect sequence homology at the nucleotide level.
[0141] Increasing nitrogen use by modifying the expression of
nitrogen assimilatory enzymes may also be a feasible approach to
enhancing yields in transgenic crop plants such as corn. The
efficiency of nitrogen use in crops is measured as enhanced yields
and is therefore an agricultural measure. This kind of adaptation
or specialization would be of no real advantage to wild type plants
which depend for their survival on a diversity of responses to
environmental conditions and not on higher yields (Sechley et al.,
1992, Int. Rev. Cyt. 134:85-163). Therefore, increases in crop
yield may be more easily realized through genetic engineering
methods such as those described herein, rather than by conventional
breeding methods.
7.0. Example
Ectopic Overexpression of Asparagine Synthetase in Plants Causes an
Increase in Plant Growth Phenotype
[0142] The following study concerns the manipulation of AS gene
expression in plants with the aim of increasing asparagine
production and testing the effects on plant growth. There are
several features of asparagine which make it preferable to
glutamine as a nitrogen transport/storage compound and hence the
increased assimilation of nitrogen into asparagine may be valuable
in vivo. Asparagine is a long-distance nitrogen transport compound
with a higher N:C ratio than glutamine. It is therefore a more
economical compound for nitrogen transport. In addition, asparagine
is more stable than glutamine and can accumulate to high levels in
vacuoles (Sieciechowicz et al., 1988, Phytochemistry 27:663-671;
Lea and Fowden, 1975, Proc. R. Soc. Lond. 192:13-26). In developing
pea leaves, asparagine is actively metabolized, but in mature
leaves that no longer need nitrogen for growth, asparagine is not
readily metabolized and is re-exported (in the phloem) from the
leaf to regions of active growth such as developing leaves and
seeds (Sieciechowicz et al., 1988, Phytochemistry 27:663-671; Ta et
al., 1984, Plant Physiol 74:822-826). AS is normally only expressed
in the dark (Tsai and Coruzzi, 1990, EMBO J. 9:323-332) therefore
35-AS1 is expressed constitutively and not only ectopically
expressed in regard to cell type, but also in regard to temporal
expression. Thus, the studies presented here examined whether the
ectopic overexpression of AS in all cell-types in a
light-independent fashion would increase asparagine production.
Also tested here was whether the increased asparagine production
provides an advantage in the nitrogen use efficiency and growth
phenotype of transgenic plants.
[0143] In addition to overexpression wild-type AS, the present
study also examined the ectopic overexpression of a modified form
of the AS enzyme (gln.DELTA.AS1) which was missing the
glutamine-binding domain. A question addressed by this study was
whether ectopic overexpression of a gln.DELTA.AS1 form of the
enzyme might produce a novel plant AS enzyme with enhanced
ammonia-dependent AS activity or whether such a mutation may have a
dominant-negative effect (Herskowitz, 1987, Nature 329:219-222) due
to co-assembly with endogenous wild-type AS subunits to form a
heterodimer (Rognes, 1975, Phytochemistry 14:1975-1982; Hongo and
Sato, 1983, Biochim et Biophys Acta 742:484-489). The analysis of
the transgenic plants which ectopically express pea AS,
demonstrated an increased accumulation of asparagine and an
improved growth phenotype (in the case of 35S-AS1), and an
increased accumulation of asparagine but accompanied by a
detrimental effect on growth phenotype (in the case of
35S-gln.DELTA.AS1). These results indicate that it is possible to
manipulate nitrogen metabolism and growth phenotype by ectopic
overexpression of AS genes. Because nitrogen is often the
rate-limiting element in plant growth and typically applied to
crops several times during the growing season, designing molecular
technologies which improve nitrogen use efficiency in crop plants
is of considerable interest to agriculture.
7.1. Materials and Methods 7.1.1. As Gene Constructs
[0144] The AS1 cDNA previously cloned from pea (Tsai and Coruzzi,
1990, EMBO J 9:323-332) was transferred from pTZ18U to the EcoRI
site of pBluescript KS- (Stratagene). A gln.DELTA.AS1 deletion
mutant was constructed using "inside-outside" PCR (Innis et al.,
1990, PCR Protocols: A guide to Methods and Applications. New York,
Academic Press pp.1-461). Coding sequence corresponding to amino
acids 2-4 (CGI) was deleted from the amino terminus of the AS1
cDNA, leaving the initiating methionine and the untranslated leader
intact. This deletion corresponded to the presumed
glutamine-binding domain of the AS enzyme comprising amino acids
MCGI which have been defined for animal AS (Pfeiffer et al., 1986,
J. Biol. Chem. 261:1914-1919; Pfeiffer et al., 1987, J. Biol. Chem.
252:11565-11570). cDNAs corresponding to wild-type AS1 and
gln.DELTA.AS1 were then transferred from pBluescript to the binary
expression vector pTEV5. This vector contains the CAMV 35S promoter
(from -941 to +26), a multiple cloning site, and the nopaline
synthase terminator. FIG. 12 shows details of the binary vector
constructions containing the AS1 cDNAs pz127 (NRRL Accession No.
B-21335) and gln.DELTA.AS1 cDNA pZ167 (NRRL Accession No. B-21336),
which were transformed into tobacco.
7.1.2. Plant Transformations
[0145] Binary vector constructions were transferred into the
disarmed Agrobacterium strain LBA4404 and subsequently into
Nicotiana tabacum SR1 using standard procedures described elsewhere
(Bevan, 1984, Nucleic Acids Res. 12:8711-8721; Horsch et al., 1985,
Science 227:1229-1231).
7.1.3. RNA Analysis of Transformants
[0146] RNA was isolated using "RNA matrix" from Bio101 and total
RNA was electrophoresed as previously described (Thomas, 1983,
Methods Enzymol. 100:255-266). Gels were capillary blotted onto
Hybond-N nylon membrane (Amersham). cDNAs were labeled using the
random primer plus extension reagent labeling system supplied by
NEN. Hybridizations were done in aqueous solution and blots were
washed in 0.1.times. SSPE, 0.1% SDS. Northern blots were probed
with the pea AS1 cDNA, pAS1 (Tsai and Coruzzi, 1990, EMBO J
9:323-332).
7.1.4. Extraction of Free Amino Acids
[0147] Tobacco leaf tissue samples were frozen in liquid nitrogen
and extracted in 10 mls of extraction media consisting of
methanol:chloroform:water (12:5:3, v/v/v). The homogenate was
centrifuged at 12,000.times. G for 15 minutes. The pellet was
extracted again and the supernatants were combined. Addition of 2.5
ml chloroform and 3.8 ml of distilled water to the supernatant
induced separation. The methanol:water phase was collected and
dried under vacuum and redissolved in 1 ml of distilled water. The
solution was filtered by passing it through a 0.45 .mu.m nylon
filter microcentrifuge tube filter system centrifuged at 12,000 g
for 2 min.
7.1.5. HPLC Determination of Amino Acid Pools
[0148] The amino acids were determined as o-phthaldialdehyde (OPA)
derivatives on a Microsorb Type O .DELTA.A Analysis column (Rainin)
using a DuPont HPLC instrument. Sample (100 .mu.L) was derivatized
with 100 .mu.l of OPA working reagent. After 2 min of
derivatization, 50 .mu.L of the derivatized sample was injected.
This gradient was produced using two eluents: A. 95% 0.1 M sodium
acetate (pH 7.2) with 4.5% methanol and 0.5% tetrahydrofluoran; B.
100% methanol. Eluents were filtered and degassed with He before
use. Detection of OPA derivatized amino acids was accomplished with
a UV spectrophotometer at 340 nm. Each determination was done twice
and the values represent the average.
7.1.6. Plant Growth Conditions
[0149] Progenies of primary transformants characterized as
expressing high levels of either as AS1 mRNA or the mutated
gln.DELTA.AS1 mRNA were germinated on MS-medium containing 100
.mu.g/ml kanamycin. After 14 days, kanamycin resistant seedlings
were transferred to 4 inch pots filled with white sand, which were
covered with saran wrap for approximately one week to prevent
excessive transpiration and enable seedlings to become established.
Pots were irrigated periodically with 1.times. Hoagland's solution
containing 10 mM potassium nitrate as the only nitrogen source.
Subsequently, between three and seven plants were sacrificed for
fresh weight determination each week, continuing for a period of
four weeks until shading of neighbors was apparent. Plants were
grown under a light-dark cycle of 16-8 h with a temperature cycle
of 24-18.degree. C. Daytime light intensity was 1000 lux.
7.2. Results
7.2.1. Construction of Transgenic Plants Expressing Pea AS1 and
GLN.DELTA.AS1
[0150] The pea AS1 cDNA (Tsai and Coruzzi, 1990, EMBO J 9:323-332)
expressed from the 35S-CaMV promoter was transferred into
transgenic tobacco (See FIG. 12 and Section 7.1 Material and
Methods) and five independent primary transformants (Z127; 1-5)
were shown to express high levels of the AS1 mRNA (see below).
Three independent transgenic lines (Z167;1-3) which contained the
AS1 CDNA with a deletion in the glutamine binding domain
(gln.DELTA.AS1) were also shown to contain high levels of transgene
RNA (see infra).
7.2.2. Northern Analysis of Transformants Expressing AS1 and
GLN.DELTA.AS1
[0151] Northern blot analysis of RNA extracted from transgenic
plants were undertaken to identify plants in which the 35S-AS1
transgene was expressed at high levels (FIG. 13). As a positive
control, RNA for AS was detected in leaves of pea plants grown in
the dark (FIG. 13, lane PL). By contrast, no AS mRNA was detected
in leaves of light-grown wild-type tobacco (FIG. 13, TL). Previous
studies have shown that tobacco AS mRNA is expressed exclusively in
tissues of plants grown the dark (Tsai and Coruzzi, 1991, Mol Cell
Biol 11:4966-4972). The transformants which overexpress AS1
(Z127-1, -3,-4, and -5) all contained high levels of AS1 mRNA, even
though these plants were grown in the light (FIG. 13). Thus, the
35S CaMV promoter produces constitutive expression of pea AS1,
whereas the endogenous AS mRNA is not expressed in tobacco leaves
in the light. The gln.DELTA.AS1 transformants also showed
constitutive high level expression of mRNA (Z167-2,-3, and -4),
compared to tobacco controls (FIG. 13). Because the AS enzyme is
notoriously unstable, the AS enzyme has never been purified to
homogeneity and antibodies for plant AS were not available for AS
protein analysis. In addition, in vitro assay detected no AS
activity due to enzyme instability.
7.2.3. Amino Acid Analysis of Transgenic Lines Expressing AS1 and
GLN.DELTA.AS1
[0152] Based on the Northern results, two independent transgenic
lines which showed high levels of AS1 mRNA (Z127-1 and Z127-4) were
selected for further analysis. Similarly, lines Z167-2 and Z167-4
were selected as high-expressing representatives of the
gln.DELTA.AS1 construction. The T2 progenies of these plants were
subjected to amino acid and growth analysis described below.
7.2.4. AS1-overexpressing Lines
[0153] Both Z127 lines selected (Z127-1 and Z127-4) showed
increased levels of asparagine (10- to 100-fold higher than
wild-type controls) (Table 4) The variation apparent among the
individual T2 plants most likely reflects the homozygosity or
heterozygosity of individuals, and the approximate 2:1 ratio of
intermediate:high asparagine levels would substantiate this
assertion. In all cases, however, a considerable increase in
asparagine is seen extending up to nearly 100-times the control
concentration. Interestingly, there is a corresponding reduction in
glutamine concentrations in these plants (although the Z127-4 data
is skewed by a single high value) and this reflects the use of
glutamine as a substrate in the AS reaction; equally predictable is
the reduction in concentration of the other substrate aspartate.
Somewhat unexpected, however, is the reduced concentration of
glutamate in the Z127 lines. From biochemical predictions and from
the data collected for the other three amino acids involved in the
AS reaction, an increase in glutamate would have been predicted.
The apparent reduction in glutamate may be the result of its high
turnover rate due to its use as a substrate in several related
processes such as transamination.
7.2.5. GLN.DELTA.AS1-overexpressing Lines
[0154] In the two lines selected which overexpress gln.DELTA.AS1,
the question assessed was whether the deletion of the
glutamine-binding domain of AS would have a dominant-negative
effect on asparagine biosynthesis. The data collected for these
lines (Z167-2 and Z167-4) is somewhat difficult to interpret due to
the variation of data values (Table 4). However, in almost every
case there is a substantial increase in asparagine concentration,
ranging from 3- to 19-fold compared to wild-type non-transgenic
tobacco. These results suggest that the transgenic lines have the
ability to accumulate asparagine with little effect on aspartate,
glutamate or glutamine pools. One possibility is that the
gln.DELTA.AS1 enzyme is able to synthesize asparagine directly from
ammonia and aspartate.
7.2.6. Plant Growth Experiment on Transformants Expressing AS1 and
GLN.DELTA.AS1
[0155] Growth analysis was undertaken using individual transgenic
T2 plants grown in white sand. These studies were aimed at
assessing growth rate under conditions which minimized interference
from neighboring plants. For this reason, fresh weight measurements
were taken only during the vegetative stage of growth (up to six
weeks post germination). During this period, plants undergo rapid
growth and are exclusively dependent on supplied nitrogen and do
not remobilize internal nitrogen sources as might occur during
bolting and flowering. Plants were grown under conditions where
nitrogen was non-limiting (i.e., regular fertilization with 10 mm
nitrate) and which reduced the photosynthetic interference of
neighboring plants. The growth analysis was terminated when such
interference became apparent. All plants analyzed were of the same
age at each time point, and analysis started at between 0.1 and 0.3
g fresh weight/plant, and continued until the plants were
approximately six weeks old when the interference of neighboring
plants became apparent and bolting was imminent.
5TABLE 4 Amino Acid Analysis in Transgenic Lines Overexpressing AS1
or gln.DELTA.AS1 PLANT ID ASN GLU GLN ASP CONTROL C 34 1399 309
1935 C 38 1425 405 1861 C 36 965 425 2015 C 47 1526 275 1720 mean
39 1335 353 1883 AS1 wild-type Z127-1-A 553 228 14 182 Z127-1-B
3399 808 60 922 Z127-1-C 213 525 81 240 Z127-1-D 487 537 17 264
Z127-1-E 3159 983 43 796 mean 1562 616 43 481 Z127-4-A 1105 838 132
451 Z127-4-B 902 2947 389 1092 Z127-4-C 373 1606 17 678 Z127-4-D
4109 691 923 1664 mean 1622 1520 365 971 gln.DELTA.AS1 Z167-2-A 684
838 352 761 Z167-2-B 1341 2947 944 3119 Z167-2-C 173 1606 1224 1946
mean 733 1797 840 1942 Z167-4-A 47 691 75 948 Z167-4-B 109 864 346
1491 Z167-4-C 137 1313 714 1705 Z167-4-D 165 1534 838 2069 mean 114
1100 493 1553 Amino acid concentrations are in nmol/gram fresh
weight
[0156]
6TABLE 5 Growth Increase of Transgenic Lines Overexpressing AS1 or
gln.DELTA.AS1 C-1 C-2 Z127-1 Z127-4 Z167-2 Z167-4 3 0.12 0.07 0.28
0.12 0.11 0.19 4 0.60 0.41 1.30 0.51 0.31 0.57 5 1.19 1.11 1.87
1.72 0.71 0.99 6 6.49 5.83 8.63 7.16 3.83 6.13 % increase at 100 90
133 110 59 94 week 6 compared to C-1 Total fresh weight means (in
grams) of transgenic lines and controls measured over a period of
three to four weeks immediately prior to the onset of bolting.
[0157] Tables 5 and 6 show the results of mean total fresh weight
determinations for lines Z127-1 and Z127-4 (overexpressing
wild-type AS1) and Z167-2 and Z167-4 (overexpressing
gln.DELTA.AS1), and these results are expressed graphically in FIG.
13. Transgenic lines overexpressing wild-type AS grew 133% and 110%
compared to control (100%) (Table 5), although in neither case was
this statistically significant when analyzed by unpaired T-test
(Table 6). Transgenic lines overexpressing the gln.DELTA.AS1
construction (Z167) did not perform comparably. The Z167-4 plants
which survived until the sixth week were indistinguishable in
growth from controls, and the Z167-2 plants which survived, were
much smaller than controls (P-0.041; significant at the 5% level)
(Tables 5 and 6, and see also FIG. 14). Comparing the three
different lines in the experiment, it was of interest that a
greater proportion of kanamycin resistant Z167 plants died.
Typically the Z167 plants were slow to germinate and looked
unhealthy when grown in pots. This was clearly reflected in the
fresh weight data collected for Z167-2, although less apparent for
the Z167-4 data, suggesting that the gln.DELTA.AS1 gene product did
indeed have a dominant-negative effect on plant growth.
7TABLE 6 Growth Increase of Transgenic Lines Overexpressing AS1 or
gln.DELTA.AS1 with Comparison to Controls By Unpaired T Test C-1
C-2 Z127-1 Z127-4 Z167-2 Z167-4 Week 6 6.49 5.83 8.63 7.16 3.83
6.13 % Increase at 100 90 133 110 59 94 week 6 compared to control
1 Number of 7 6 7 7 3 5 Plants (week 6) Standard 0.60 1.07 1.15
0.88 0.92 0.85 Error Standard 1.58 2.61 3.05 2.34 1.60 1.89
Deviation "T" for 1.65 0.63 2.43 0.35 unpaired test (12) (12) (8)
(10) to control-1 (df) Probability 0.125 0.54 0.041 0.731
Significance NS NS * NS Total fresh weight means for transgenic
lines (in grams) and controls at week 6. The statistical analysis
was done for the final week's measurement only and control-1 was
selected for the T-test df - degrees of freedom; The probability of
the populations being related was deemed to be significant (*) for
P < 0.05; NS - not significant
7.3. Discussion
[0158] Reported here are studies in which AS is ectopically
overexpressed in transgenic plants to test the effects of this
manipulation on primary nitrogen assimilation and on plant growth.
In particular, the cell-specific expression pattern of AS were
altered and the regulation of AS with regard to light was also
modified. In wild-type plants, AS is normally only expressed in the
phloem (Tsai, 1991, Molecular Biology Studies of the
Light-Repressed and Organ-Specific Expression of Plant Asparagine
Synthetase Genes. Ph.D. Thesis, The Rockefeller University, New
York, NY), and its expression is dramatically repressed by light in
both photosynthetic and non-photosynthetic tissues (Tsai and
Coruzzi, 1990, EMBO J 9:323-332; Tsai and Coruzzi, 1991, Mol Cell
Biol 11:4966-4972). Here, the wild-type AS1 of pea and a mutated
form of AS1 (gln.DELTA.AS1) were expressed under the control of a
constitutive promoter (35S-CaMV) in transgenic tobacco so that AS1
is expressed in all cell types, in a light-independent fashion. The
physiological significance of constitutively expressing AS1 in
cells where it is not normally expressed may have considerable
impact on plant nitrogen metabolism. For example, asparagine is
involved in photorespiratory nitrogen recycling (Givan et al.,
1988, TIBS 13:433-437; Ta et al., 1984, Plant Physiol 74:822-826),
thus the ectopic expression of AS in photosynthetic cells may have
dramatic impact on photorespiration. Furthermore, the expression of
an ammonia dependent AS enzyme in this context may aid in the
reassimilation of photorespiratory ammonia.
[0159] Four independent transgenic tobacco lines expressing 35S-AS1
have been shown to express a wild-type pea AS1 transgene
constitutively. Two lines were analyzed further (Zl27-1 and Z127-4)
and it was shown that the expressed AS1 gene was functional since
free asparagine accumulated to high levels in transgenic leaf
tissue; typically transgenic lines Z127-l and Z127-4 accumulated
between 10- and 100-fold more asparagine than control untransformed
tobacco lines. These increased asparagine levels were predictably
accompanied by a reduction in the AS substrates, glutamine and
aspartate. However, it may still be possible to channel more
inorganic nitrogen into the nitrogen transport compound asparagine
by providing higher endogenous levels of glutamine, a substrate for
AS.
[0160] The plant growth experiment on the Z127 transgenic plants
was aimed at determining whether the accumulation of asparagine in
the AS1 overexpressing plants might have a positive effect on
growth during the vegetative stage of plant development. The rapid
leaf development which occurs during vegetative growth imposes a
strong demand for nutrient availability and nitrogen is typically
the most critical nutrient at this time due to the synthesis of new
proteins in expanding and enlarging tissues. Nitrogen assimilated
and accumulated at this time is subsequently recycled in the plant
and deposited in seed reserves; as well as being a major
long-distance transport amino acid, asparagine also plays an
important role in the formation of seed reserves (Dilworth and
Dure, 1978, Plant Physiol 61:698-702; Sieciechowicz et al., 1988,
Phytochemistry 27:663-671). The two Z127 lines were found to
outgrow untransformed controls over a six week period up to the end
of vegetative growth and conferred a 10% and a 33% growth
advantage. However, these figures were not statistically
significant when a T-test is performed. Thus, although the plants
make 10- to 100-fold higher levels of asparagine, it is possible
that glutamine levels are limiting relative to increases in growth.
Also presented here is the finding that overexpressing GS in
transgenic tobacco can confer a greater growth advantage which is
statistically significant (supra). As glutamine is a substrate for
asparagine biosynthesis both are pivotal amino acids in the primary
assimilation of inorganic nitrogen. It can therefore be anticipated
that creating transgenic lines which express both GS and AS at high
levels (by crossing AS and GS overexpressers) may have even more
advantageous growth traits than either parent. In particular, the
approaches disclosed here have the advantage that assimilation in
transgenic lines will not be restricted to a few cell types,
enabling available nitrogen in all plant cells to be utilized. The
ectopic overexpression of both GS and AS in a single plant may have
the advantage of avoiding glutamine accumulation; since glutamine
is an active metabolite in the presence of high concentrations of
glutamine may upset cell metabolism. By contrast, asparagine is a
relatively inert compound able to store nitrogen economically. In
addition, asparagine is formed in a reaction which liberates a
molecule of glutamate then available to accept a further unit of
ammonia (Lea and Fowden, 1975, Proc. R. Soc. Lond. 192:13-26).
[0161] In addition to the ectopic overexpression of wild-type AS,
the plant glutamine-dependent AS was modified in an attempt to
enhance its ammonia-dependent activity. In particular, it has been
shown in animals that antibodies to the glutamine-binding domain of
AS inhibit glutamine-dependent AS activity present on the same AS
polypeptide, yet enhance the ammonia-dependent activity (Pfeiffer
et al., 1986, J. Biol. Chem. 261:1914-1919; Pfeiffer et al., 1987,
J. Biol. Chem. 252:11565-11570). By analogy, a site-specific mutant
was created in a pea AS1 cDNA (Tsai and Coruzzi, 1990, EMBO J
9:323-332) which mutation specifically deleted the three amino
acids required for glutamine binding (gln.DELTA.AS1). By
introducing this glnS.DELTA.AS1 into transgenic plants, it might be
possible to enhance the ammonia-dependent AS activity and/or
inhibit the endogenous glutamine-dependent AS activity through
subunit poisoning and the formation of heterodimers of wild-type
and mutant subunits. Two independent transgenic lines, Z167-2 and
Z167-4, which overexpress the gln.DELTA.AS1 transgene were found to
be capable of accumulating asparagine levels approximately 3- to
19-times greater than untransformed tobacco controls. The activity
of the gln.DELTA.AS1 gene in assimilating asparagine is suggestive
of the modified enzyme having the capability of utilizing a
nitrogen substrate other than glutamine (e.g., ammonia). By analogy
to the known ammonia-dependent AS activities of the E.coli AsnA
gene and mammalian AS, the high levels of asparagine in the
transgenic plants which express the mutated plant gln.DELTA.AS1
enzyme suggest that the gln.DELTA.AS1 enzyme can assimilate ammonia
directly into asparagine and therefore bypass GS in primary
nitrogen assimilation. If this suggestion is correct, it is also
apparent that the gln.DELTA.AS1 gene is not as efficient in
synthesizing asparagine as the overexpressed wild-type AS1, based
on the relative levels of asparagine in these transgenic plants
(Z167 vs. Z127).
[0162] Transgenic lines expressing gln.DELTA.AS1 (Z167-2 and
Z167-4) did not outgrow untransformed controls; indeed they
typically grew more poorly than untransformed plants as evidenced
by the performance of Z167-2 and the higher proportion of Z167
plants to die before the end of the experiment. It is curious that
these plants should accumulate 3- to 19-fold higher levels of
asparagine in their leaves, yet grow more poorly. Plant AS is
believed to assemble as a homodimer (Rognes, 1975, Phytochemistry,
14:1975-1982). In leaf mesophyll tissue where wild-type AS is not
normally expressed, the glni.DELTA.AS1 form is able to
self-assemble into homodimers and form an enzyme capable of
generating asparagine. In phloem cells, however, gln.DELTA.AS1
subunits may co-assemble with wild-type AS subunits, thereby
inactivating wild-type AS as a dominant-negative mutation
(Herkowitz, 1987, Nature 329:219-222). In the gln.DELTA.AS1 plants,
asparagine synthesized in leaf mesophyll cells may be unable to be
transported to and loaded into the phloem and this could account
for the poor growth phenotype of these transgenic lines. These
observations highlight the specialization of cell-type function,
and cell-specific gene expression of nitrogen metabolic genes and
their impact on plant nitrogen metabolism.
8. Deposit of Microorganism
[0163] The following microorganism are deposited with the
Agricultural Research Culture Collection, Northern Regional
Research Center (NRRL), Peoria, Illinois and are assigned the
following accession numbers:
8 Strain Plasmid NRRL Accession No. Escherichia coli, Z3 pZ3
B-21330 Escherichia coli, Z9 pZ9 B-21331 Escherichia coli, Z17 pZ17
B-21332 Escherichia coli, Z41 pZ41 B-21333 Escherichia coli, Z54
pZ54 B-21334 Escherichia coli, Z127 pZ127 B-21335 Escherichia coli,
Z167 pZ167 B-21336
[0164] Although the invention is described in detail with reference
to specific embodiments thereof, it will be understood that
variations which are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0165] Various publication are cited herein, the disclosure of
which are incorporated by reference in their entireties.
* * * * *