Transgenic plants expressing a prokaryotic ammonium dependent asparagine synthetase

The gene asnA which encodes a prokaryotic ammonium-specific asparagine synthetase (ASN-A) can be introduced into plant cells. Such transformed cells and plants developed therefrom not only tolerate glutamine synthetase inhibitors but are effectively stimulated by such herbicides.

FIELD OF THE INVENTION 
The invention relates to transgenic plants expressing a prokaryotic 
ammonium dependent asparagine synthetase. 
BACKGROUND OF THE INVENTION 
Asparagine plays an important role as a transport form of nitrogen and in 
many plants--including nitrogen fixers--it is the principle compound 
involved in the transfer of nitrogen from the roots into the transpiration 
stream. In plants asparagine is formed from glutamine, aspartate and ATP 
catalyzed by the asparagine synthetase ASN (E.C. 6.3.5.4 ) whereby 
glutamate, AMP and pyrophosphate are formed as by-products. 
SUMMARY OF THE INVENTION 
It now has been found that the prokaryotic, ammonium dependent asparagine 
synthetase ASN-A (E. C. 6.3.1.1) can be introduced into plant cells, 
resulting in transgenic plants which show a number of advantages: The 
plants show a more efficient net photosynthetic CO.sub.2 -fixation, an 
increased growth rate, accelerated plant development, earlier flower 
formation, increased green mass and plant dry weight. Thus, the growth 
area can be extended to regions with a less favourable climate and in 
regions with a warm climate e.g. three instead of two crops are possible. 
Furthermore, the transgenic plants tolerate the application of glutamine 
synthetase (GS) inhibitors, e.g. phosphinothricine (PPT) or methionine 
sulfoximine (MSX) and even show a stimulation of photosynthesis and growth 
upon application of such inhibitors.

DETAILED DESCRIPTION 
In contrast to the ASN encoding gene (or genes) of higher plants (asn gene) 
the asnA gene of E. coli codes for a different type of ASN which uses 
ammonium rather than glutamine for the production of asparagine (Cedar and 
Schwartz (1969) J. Biol. Chem. 244, 4112-4121). The gene for this enzyme 
has been isolated and characterized by Nakamura et al. (1981) Nucleic 
Acids Research 9, 4669-4676. It was found that this prokaryotic enzyme is 
active in plants and it opens a new ammonium assimilation pathway in these 
transgenic plants. It results in an overall change of the plant nitrogen 
metabolism with a stimulatory effect on growth and green mass production. 
Under normal conditions in the transformants both GS and ASN-A use ammonia. 
The advantage of the bacterial pathway will be more pronounced during 
darkness when the chloroplast GS activity is limited by the reduced 
availability of ATP, energy charge and magnesium ions (O'Neal and Joy 
(1974) Plant Phys. 54, 773-779; Joy (1988) Can. J. Bot. 66, 2103-2109). 
Moreover, the expression of the bacterial asn-A gene in plants even allows 
an assimilation of ammonia when the plant GS is blocked by specific 
inhibitors like PPT. In non-transformed plants the inhibition of GS 
activity disturbs the main road of ammonia utilization and the 
accumulation of ammonium is one of the key factors in lethality of the 
treated plants (Tachibana et al. (1986) J. Pesticide Sci. 11, 33-37). Thus 
the presence of the bacterial enzyme can reduce the ammonia accumulation 
in PPT-treated transgenic plants which so not only can survive doses of 
herbicides which are lethal for wild-type plants but rather the so treated 
transgenic plant shows growth stimulation. 
It is apparent for a skilled person that these positive effects are not 
limited to the asnA gene from E. coli since other bacteria contain the 
same gene or a gene having the same capability of amidating asparaginic 
acid and its salts to yield asparagine. 
Thus the invention relates to the use of a prokaryotic asnA gene in a plant 
cell, a gene construct comprising a gene encoding a prokaryotic asnA, 
operatively linked to a regulatory sequence which effects the expression 
of the said gene in a plant cell, a vector containing such a gene 
construct, a plant cell transformed with such a gene construct or vector 
and expressing a prokaryotic ammonia specific asparagine synthetase in a 
plant, especially a crop plant, and seeds or propagation material of such 
plants which contain transformed cells as hereinbefore defined. 
Preferred embodiments comprise the use of the E. coli asnA gene encoding 
the said enzyme and synthetic genes encoding the said enzyme, especially 
genes comprising codons which are preferably used by plants. The invention 
also comprises genes which encode enzymes having a different amino acid 
composition than the natural enzymes but with essentially the same 
catalytic activity by deleting or adding codons or by replacing codons in 
the natural genes by such which encode a different amino acid. All such 
modifications are within the ordinary skill of persons involved in this 
art. 
EXAMPLES 
Example I 
Expression of the E. coli asnA gene with the RUBISCO small subunit promoter 
in tobacco 
1. Production of transgenic tobacco plants 
Based on the complete nucleotide sequence of the asnA gene from E. coli 
(Nakamura et al., (1981) Nucleic Acids Research 18, 4673, FIG. 3) we 
recloned the PstI-HgaI fragment from the plasmid pMY114 into pUC9. Then 
the asnA gene (1.1 kb) was linked to the promoter of the small subunit 
gene for pea ribulose 1,5-bisphosphate carboxylase ("RUBISCO", 
Herrera-Estrella et al. (1984) Nature 310, 115-120) and the whole fragment 
was introduced into the Agrobacterium vector pPCV001 (Koncz and Schell 
(1986) Mol. Gen. Genet. 204, 383-396). After leaf disc transformation of 
SRI tobacco plants the transgenic plants were identified on the basis of 
their kanamycin resistance. Among several transformants we selected two 
plants (ASP4, ASP5) which showed tolerance against treatment with 1 kg/ha 
PPT. As result of this PPT treatment the SRI control plants were 
completely killed and we could never find outgrowings with a capability 
for flowering and seed production. The ASP4 and ASP5 transformants showed 
symptoms only on the lower and older leaves while the meristematic region 
could overcome the inhibition. After continuation of growth these plants 
flowered and produced seeds. 
Selfing the ASP4 and ASP5 transgenic tobacco plants has resulted in a 
segregating seedling population with resistant and sensitive sexual 
progenies. Under the in vitro conditions used the presence of 10 .mu.M 
L-PPT in the culture medium could clearly discriminate between the two 
phenotypes. 
The presence of the asnA sequence in the genome of the transformants was 
also shown by Southern DNA hybridization. After digestion of plant DNAs 
with EcoRI a hybridizing fragment was revealed in the transformed plants. 
In Northern hybridization analysis, a low amount of mRNA which was 
homologous to the asnA gene was detected in the total RNA isolated from 
the in vitro grown ASP5 transformant. 
2. Reduced ammonia accumulation in transgenic tobacco plants 
The inhibition of GS activity by PPT treatment causes a rapid increase in 
ammonia concentration in leaves of control tobacco plants. The rate of 
ammonia accumulation measured with the microdiffusion method and 
subsequent nesslerization (Shelp et al. (1985) Can. J. Bot. 63, 1135-1140) 
depends on the concentration of the applied herbicide. 
At a dose of 0.5 kg/ha the transgenic plants can overcome the effects of 
PPT treatment (Tab. 1). 
TABLE 1 
______________________________________ 
Accumulation of ammonia in control tobacco plants (SR1) and 
in transgenic plants with the asnA gene after spraying with 
0.5 kg/ha PPT 
ammonia concentration (mM) 
hours SR1 ASP4 ASP5 
______________________________________ 
up to 4 0.58 0.42 0.40 
6 1.20 0.82 0.78 
24 1.70 0.70 0.75 
48 1.95 0.60 0.50 
______________________________________ 
A reduced level of accumulation can also be seen in these plants in 
comparison to SR1 tobacco plants after spraying with 1 kg/ha (Tab. 2). The 
detected lower ammonia level will be responsible for the less pronounced 
damage of transformed plants. 
TABLE 2 
______________________________________ 
Effects of 1 kg/ha PPT on ammonia concentration in tobacco 
(SR1) and transgenic plants (ASP4, ASP5) 
ammonia concentration (mM) 
hours SR1 ASP4 ASP5 
______________________________________ 
up to 6 0.8 0.78 0.88 
8 2.2 0.87 0.95 
24 4.9 2.0 1.40 
48 8.6 4.1 3.6 
______________________________________ 
3. Stimulation of plant growth and development 
Detailed comparison of growth behaviour between control and ASP plants 
revealed considerable differences: An increased growth rate was 
characteristic for the transgenic plants but a more significant 
stimulation was achieved by treatment of ASP plants with low doses of PPT. 
The basic as well as the PPT induced acceleration in growth could be 
demonstrated by various types of growth curves. FIG. 1 shows that while 
the spraying with 0.025 kg/ha PPT already inhibited the growth of SR1 
plants a large stimulation was detected in both of the transformants. 
Spraying with 0.05 kg/ha PPT has a negative influence on all plants. Each 
point represents the average height of three plants. 
The differences between the various lines under control and treated 
conditions are also detectable if we characterize the growth of plants by 
Baule Mitscherlich-curves (FIG. 2) under greenhouse conditions. The 
inhibition and stimulation of growth can be followed by the slope of plots 
with characteristic alpha angles shown in FIG. 2. 
4. Increased dry weight in transgenic plants 
In addition to the differences in plant height the stimulatory effects were 
also detectable by measuring dry weight. The data shown in Table 3 
demonstrate the higher productivity of the asnA transformants: 
TABLE 3 
______________________________________ 
Final dry weight (gr) of control (SR1) and transformants 
(ASP4, ASP5) 
Treatment 
Lines Control 0.025 kg/ha PPT 
______________________________________ 
SR1 3.19 100% 2.76 100% 
ASP4 3.85 120% 4.79 173% 
ASP5 3.74 117% 5.05 183% 
______________________________________ 
Example II 
Effect of the asnA gene driven by the CaMV35S promoter in transgenic plants 
1. Selection of transgenic plants 
As an alternative approach we have introduced plasmid molecules (pUC) 
carrying the E. coli asnA gene with the CaMV35S promoter into SR1 leaf 
protoplasts by direct DNA uptake (R. X. Fang et al. (1989) The Plant Cell 
1, 141-150). The transformants were directly selected on the basis of 
their PPT resistance. Plants were regenerated from micro calli grown in 
the presence of 10 .mu.M L-PPT. The Southern hybridization confirmed the 
presence of the asnA gene in DNA isolated from the PPT resistant 
regenerants. 
2. Reduced ammonia accumulation and improved PPT tolerance in transgenic 
plants 
Selling of regenerated transformants resulted in segregating progenies with 
various levels of PPT resistance (medium supplemented with up to 30 .mu.M 
L-PPT). In agreement with the resistant phenotype the transformed plants 
accumulate less ammonia than the SR1 plants when sprayed with 1 kg/ha PPT 
(Tab. 4). 
TABLE 4 
______________________________________ 
Ammonia accumulation after spraying the plants with 1 kg/ha 
PPT 
ammonia concentration (mM) 
hours SR1 ASP70 ASP95 
______________________________________ 
6 6.85 2.92 1.95 
24 9.50 5.60 5.10 
48 22.3 13.60 17.40 
120 58.60 28.30 35.6 
144 113.00 39.40 50.00 
______________________________________ 
3. Efficiency of photosynthesis 
Both the control SR1 and transgenic tobacco plants were characterized by 
various parameters of photosynthesis such as the CO.sub.2 fixation rate 
(Szajko et al. 1971, Acta Agr. Acad. Hung. 20, 247-260) and fluorescence 
induction (Hideg et al., 1986, Photobiochem. Photobiophys. 12, 221-230). 
Under greenhouse conditions the plants were treated with various doses of 
PPT and the content of ammonium was also determined. As shown by Table 5 
the transgenic plants with the ASN-A gene exhibit a considerable increase 
in efficiency of net CO.sub.2 fixation in comparison to the control 
plants. Application of low dose PPT treatment can further stimulate 
CO.sub.2 fixation, while the difference between SR1 plants with or without 
PPT (50 g/ha) treatment is not statistically significant. The Table 5 
provides also evidence that in the case of tobacco plants the inhibitory 
concentration of PPT causes ammonium-accumulation with serious damage in 
photosynthesis by inhibition of electron transport and a 50% reduction of 
CO.sub.2 fixation. Under the same conditions the transformed plants (ASP 
70) can tolerate the treatment as the photosynthetic function is 
concerned. 
TABLE 5 
__________________________________________________________________________ 
Parameters of photosynthesis 
PPT Ammonia 
CO.sub.2 Fluorescence 
treat- concen- 
fixation induction 
ment tration 
(.mu.mol CO.sub.2 /dm.sup.2 xh) 
(in % of control SRI) 
Lines 
(g/ha) 
(mM) x .+-.s.sub.x 
n P 1% 
F.sub.m 
F.sub.o 
F.sub.i - F.sub.o /F.sub.m 
__________________________________________________________________________ 
- F.sub.o 
SR1 0 0.37 38.17 
12.05 
20 
- 100 
100 
0.45 
50 2.00 44.23 
17.73 
20 
- 101 
102 
0.44 
750 34.35 
19.54 
12.42 
20 
+ 80 194 
0.59 
ASP70 
0 0.47 49.32 
7.86 
20 
+ 98 114 
0.38 
50 2.70 58.07 
6.06 
20 
+ 99 110 
0.42 
750 15.80 
31.61 
14.16 
20 
- 92 144 
0.49 
__________________________________________________________________________ 
The analysis was carried out 4 days after PPT treatment. 
4. Growth behaviour of asnA transformant plants 
Analysis of growth rate (mm/day) reproducibly showed accelerated growth of 
transformants during the early plant development. Data are shown in Table 
6 for plants grown in the green house. 
TABLE 6 
__________________________________________________________________________ 
Growth rate (mm/day) during various periods of plant 
development (green house) 
Periods (6 days) Final plant height 
Lines I II III 
IV V VI VIII 
VIII 
(cm) 
__________________________________________________________________________ 
SR1 0.29 
0.45 
0.27 
0.52 
0.75 
1.31 
1.93 
1.37 
41.08 
Asp70/1 
0.60 
1.15 
0.43 
1.23 
1.83 
2.08 
1.53 
0.50 
58.0 
141% 
Asp70/2 
0.61 
0.93 
0.50 
0.75 
1.18 
1.51 
1.83 
0.25 
48.5 
118% 
__________________________________________________________________________ 
SR1: average of 5 plants 
ASP70/1 and ASP70/2: individual plants 
The analysis of these plants under field conditions revealed similar 
differences as it was observed in the green house (Table 7). The growth 
rate of ASP plants during period I-III was considerably higher than in the 
case of SR1 plants. In this experiment the stimulatory effect of PPT on 
the transgenic plants was also confirmed especially in the last growing 
period. The Baule-Mitscherlich curves (FIG. 3) clearly demonstrate that 
the ASP plants exhibit faster growth than control SR1 plants grown in the 
field. 
TABLE 7 
______________________________________ 
Growth rate (mm/day) during various periods of plant 
development (field experiment) 
Treat- Periods (7 days) 
Final plant 
ment Lines I II III IV height (cm) 
______________________________________ 
Control SR1 0.36 0.85 1.31 3.24 42.5 100% 
Asp70 0.45 1.07 1.50 3.14 47.6 112% 
Asp95 0.48 1.14 1.92 3.24 51.5 121% 
25 g/ha SR1 0.29 0.56 1.15 2.74 35.0 100% 
PPT Asp70 0.44 1.02 1.69 3.84 53.8 154% 
Asp95 0.31 0.88 1.27 3.78 48.7 139% 
______________________________________ 
Average of 5 plants 
5. Productivity of asnA transformants 
As shown by Table 8 the total green mass as well as the dry weight was 
increased in ASP plants in comparison to SR1 plants. Here we can also see 
that the transgenic plants are significantly stimulated by PPT treatment. 
At the same time the control SR1 plants are already inhibited by the 
spraying. 
TABLE 8 
______________________________________ 
Control 25 g/ha PPT 
Lines Total % Leaf % Total % Leaf % 
______________________________________ 
Green mass (gr) 
field test 
SR1 86.5 100 57.4 100 78.7 100 43.4 100 
ASP70 95.2 110 62.3 108 139.9 178 92.41 
213 
ASP95 103.8 120 68.4 119 105.0 133 71.56 
165 
Dry weight (gr) 
field test 
SR1 6.66 100 4.83 100 6.42 100 5.05 100 
ASP70 8.05 121 5.82 120 10.99 171 8.56 169 
ASP95 8.48 127 6.34 131 8.96 140 6.78 134 
______________________________________ 
All average data from 5 plants