- Email: [email protected]
Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory
Research Paper
EAEF 2(3) : 83-88, 2009
Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory Tsuyoshi OKAYAMA*1, Kenichi OKAMURA*1, Haruhiko MURASE*2 Abstract Leaf lettuce (Lactuca sativa L. cv. ‘Greenwave’) containing an introduced Cauliflower mosaic virus-35S promoter fused onto the the
-glucuronidase (GUS) gene was grown in a plant factory for 50 days from seeding.
Measurements of the GUS expression level of leaves indicated that the GUS protein that accumulated in the leaves was very stable and did not degrade. Consequently, the GUS expression per plant increased exponentially as the cultivation period became longer. This stability of the recombinant protein indicates that a longer cultivation period would be more efficient than the conventional cultivation period in a plant factory. However, the optimum cultivation period depends on the stability of the recombinant protein which is required. [Keywords] CaMV-35S promoter, -glucuronidase, leaf lettuce, plant factory, transgenic
I
Introduction
Since 1989 when the antibody from mouse was expressed in transgenic tobacco by Hiatt et al. (1989), transgenic plants have been considered promising systems for producing useful protein in bulk amounts at relatively low cost (Smith, 1996; Daniell et al., 2001; Streatfield et al., 2001). If useful proteins for oral administration, such as vaccines, can be produced in edible plants, the purification procedures can be eliminated (Daniell et al., 2001). Lettuce is one of the most popular vegetables in the world, and has already been examined as a host plant for heterologous protein production (Torres et al., 1993; Kapusta et al., 1999; Joh et al., 2005; Negrouk et al., 2005; Kim et al., 2006; Sun et al., 2006; Kim et al., 2007; Li et al., 2007). Lettuce is cultivated commercially in many plant factories in which environmental conditions such as air temperature, relative humidity, light intensity and CO2 concentration are controlled precisely (Morimoto et al., 1995; Murase, 2002). Lettuce has a short cultivation cycle from germination to harvest, and requires relatively low light intensity. In addition, the environment for cultivating lettuce is easy to control, because lettuce is usually cultivated during only its vegetative growth stage. These advantages are important for exploring the relationship between environmental growth conditions and heterologous protein production in a short research period.
When a transgenic lettuce plant is used as a host plant to produce heterologous proteins, most of the proteins will be stored in leaves. Therefore, maximization of total leaf fresh weight seems to be the optimum way to maximize the amount of the heterologous protein. However, the amount of proteins in a leaf decreases because of senescence and environmental stress (e.g. drought, temperature, shading, etc.) (Smart, 1994). Stevens et al. (2000) investigated the relationship among leaf senescence, environmental conditions (temperature and light intensity) and the amount of antibodies (IgG) expressed in tobacco leaves. They reported that the top (young) leaves contained about twice the amount of antibodies as the base (old) leaves. This indicates that the maximization of leaf fresh weight does not equate to the maximization of the amount of heterologous protein in the plant. Smith (1996) noted that the way in which heterologous protein is accumulated in plants needs to be revealed before the technology can be used commercially. Therefore, a new comprehensive criterion is required for maximizing the amount of heterologous protein in plants. Okayama et al. (2008) developed a model for simulating heterologous protein production in lettuce. The model, which focused on the time course of fresh weight and protein concentration of each leaf, indicated the existence of an optimum
cultivation
cycle
and
the
advantages
and
disadvantages of protein production at low and high
*1 JSAM Member, Corresponding author, Osaka Branch, Okayama Lab., NISSHOKU Corporation, 1-2 Naka-ku, Sakai, Osaka 599-8570, Japan; [email protected] *2 JSAM Member, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-1 Naka-ku, Sakai, Osaka 599-8531, Japan
84
Engineering in Agriculture, Environment and Food Vol. 2, No. 3 (2009)
temperatures. In that study, it was assumed that a leaf with a
from lettuce leaves using about 20 mg fresh weight of tissue
higher concentration of total soluble protein (TSP) would be
ground in 250 ul extraction buffer consisting of 50 mM
able to accumulate a higher amount of heterologous protein.
sodium phosphate at pH 7.0, 10 mM EDTA, 0.1% Triton
However, the production and degradation process of
X-100,
heterologous protein appears to be much different from the
2-mercaptoethanol. The homogenate was centrifuged for 5
processes of TSP. Therefore, the heterologous protein should
min at 12,000 rpm at 4 ºC. A soluble extract was used to
be used as a parameter for more practical modelling.
determine GUS activity with 1 mM 4-methylumbelliferyl
0.1%
sodium
lauryl
sarcosyl,
and
10
mM
Although there are many reports about the expression of
- -D-glucuronide as a substrate. The reaction was incubated
heterologous protein in transgenic plants, the cultivation
at 37 ºC for 60 min, and 100 μl aliquot was removed and 2 ml
periods in the most of them were only a few weeks and the
0.2 M Na2CO3 was added to stop the enzyme reaction. The
changes in the amount of heterologous protein per plant over
reaction product 4-methylumbelliferone (MU) was detected
time were not mentioned. Therefore, in this study, transgenic
with a fluorescence spectrophotometer (F-7000, Toshiba Co.,
lettuce plants containing an introduced cauliflower mosaic
Ltd, Japan) at 365 nm excitation/460 nm emission. This GUS
virus (CaMV)-35S promoter fused to the
activity (nmol (MU) min-1) was used for expressing the
-glucuronidase
(GUS) gene were grown in a plant factory which had a
quantitative accumulation of the GUS protein.
conventional cultivation system, and the time course of
TSP concentrations of plant extracts were determined by the
accumulation of heterologous protein in leaves and plants was
dye-biding method of Bradford (1976) with a kit supplied by
investigated to collect information for establishing a practical
Bio-Rad.
heterologous protein production system.
Leaf fresh weight and GUS activity were measured on 25, 30, 34, 40, and 50 d after seeding. Five samples were
II 1.
Materials and Methods
measured on 25, 30 and 34 d and three samples were measured on 40 and 50 d.
Transgenic lettuce
Leaf lettuce (Lactuca sativa L. cv. ‘Greenwave’) was used as the host plant. The binary vector was pBI121 (Clonetech. Inc., USA) containing the neomycine phosphotrasferase II (NPTII) gene, conferring kanamycin resistance, and the GUS
III
Results and Discussion
In this study, leaves were numbered from a cotyledon (Fig. 1).
...
gene containing a functional intron within its coding region under the control of the CaMV-35S promoter. Primary transformants (T0 generation) were self-pollinated and their T1 progenies were self-pollinated to obtain T2 seeds in a closed growth chamber. The seeds were used in the experiment. The percentage of seed germination was consistently over 90%. 2.
Environmental conditions
The transgenic lettuce plants were grown in the plant factory at Osaka Prefecture University, Osaka, Japan. The photo/dark cycle was 14/10 h d-1. Light intensity was approximately 250 μmol m-2 s-1 (photosynthetic photon flux density) and was provided by fluorescent lamps. A half-strength nutrient solution (A-type Otsuka formula, Otsuka Chemical Co., Ltd., Japan) at pH 6.0, electric conductivity of 0.12 S m-1, was supplied to the plants using the deep flow technique. CO2 level was maintained at approximately 1000 μmol mol-1. Air temperature and relative humidity were kept at 23/18 ºC and 50/80% (photo/dark period), respectively. 3. Extraction and GUS assay Quantitative evaluations of GUS expression were carried out following Jefferson et al. (1987). Extracts were prepared
No.8
No.7
No.6
No.5 No.3 No.1
No.4 No.2
Fig. 1 Leaf position and numbering. Nos. 1 and 2 were cotyledons. 1. Relation between GUS activity and TSP The GUS activity and TSP per gram fresh weight (gFW) was measured in 10, 11, 12, and 13 d-old leaves (Fig. 2). The photosynthetic activity becomes the maximum before the leaf expands fully (Hodanova, 1981). Therefore, the protein involved with photosynthesis declines progressively, due to a general decrease in synthesis and an increase in degradation by proteases, and is mobilized to the rest of the plant (Smart, 1994). Our results show that the TSP concentration gradually decreased as a leaf aged. However, the GUS protein kept increasing. This indicates that there is less relation between the respective amounts of TSP and GUS protein and agrees with the high stability of the GUS protein reported by
85
OKAYAMA, OKAMURA, MURASE: Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory Jefferson et al. (1987).
Fwi
Fw maxi Fw max i rgri ( tgri t ) 1 e 1 e rseni (tseni t )
(1)
where Fwmax = maximum leaf fresh weight (g); rgr = 8 GUS activity -1 -1 (nmol min gFW )
7 6 5 4 3 2
GUS activity
1
TSP conc.
0 10
Fig. 2
11 12 Leaf age (d)
growth speed (d-1); tgr = related to onset of growth (d); rsen =
13
Changes in GUS activity and total soluble protein
concentration in a leaf as it ages. Error bars indicate the
senescence (d-1). 14 Leaf Leaf Leaf Leaf
12 10 8
No.5 No.7 No.9 No.11
6 4 2 0 15
standard deviation (n=5). 1.
senescence speed (d-1); and tsen = related to onset of
Leaf fresh weight (g)
10 9 8 7 6 5 4 3 2 1 0
Total soluble concentration -1 (mg gFW )
9
25
35
45
55
Day after seeding
Fig. 4 Time course of fresh weight of leaves No.5 to 11.
Leaf fresh weight
The number of leaves per plant was 8, 10, 18, and 28 on 25,
Bars indicate standard deviations (n=5 on 25, 30 and 34 d; n=3 on 40 and 50 d).
30, 40, and 50 d, respectively (Fig. 3 left). The total leaf fresh weight per plant exponentially increased and more than doubled from 40 d (69.8 ± 11.9 g) to 50 d (190.2 ± 41.6 g)
Leaves in the higher positions grew faster and had higher
(Fig. 3 right). Leaves from No.1 to 4 perished by 40 d, and
fresh leaf weight than leaves in the lower positions (Table 1).
leaves No. 5 and 6 perished by 50 d. The reason of the
When a leaf was shaded by other leaves above it, its fresh
perishing was senescence with aging and shading by higher
weight decreased because of senescence.
leaves (Ackerly, 1999). Table 1 25 d 30 d 40 d 50 d
16 Leaf fresh weight (g)
Total leaf fresh weight per plant (g)
18 14 12 10 8 6 4 2 0 0
5
10
15
20
Leaf Number
25
30
Parameters in equation (1) relating to changes in leaf fresh weight.
250
Leaf No.5 Leaf No.7 Leaf No.9 Leaf No.11
200 150 100
Fw max 0.8 3.9 8.8 9.5
rgr 0.51 0.52 0.69 0.61
tgr 23.6 29.9 33.4 36.2
rsen 0.85 1.12 0.60 -
tsen 40.48 50.32 51.73 -
50 0 25 d
30 d
40 d
50 d
Day after seeding
2. GUS activity The GUS activity was not uniform within the leaves (Fig.5); it increased exponentially from the base to the tip.
Fig. 3 Leaf fresh weight (left) and total leaf fresh weight
This is because the tip developed earlier than the base.
per plant (right), bars indicate standard deviations (n=5 on 25
Therefore, the period for production of GUS protein in the
and 30 d; n=3 on 40 and 50 d).
acral cells was longer than that in the basal cells. In the following GUS activity measurement, the sample was
Figure 4 focuses on the time course of the fresh weight of
extracted from the middle of a leaf, because the GUS activity
leaves No. 5, 7, 9 and 11. Leaf No. 5 perished by 40 d, and the
in the middle part represents approximately the mean value of
color of leaf No. 7 was yellow (almost perished) on 50 d. In
the GUS activity in a whole leaf.
order to evaluate these data objectively, the time course of leaf fresh weight (Fw) (g) of leaf No. i at growth time (t) (d) can be expressed with the following equation including two logistic functions (Okayama et al., 2008). One logistic function describes the growth and the other describes senescence:
86
Engineering in Agriculture, Environment and Food Vol. 2, No. 3 (2009) activity of other leaves was nearly saturated after 40 d.
-1
50
0 00
mm
y = 0.1238e R2 = 0.7187
90 -1
6
-1
5 4
-1
GUS activity (nmol min-1 gFW-1)
mm
100
0.0294x
7
3 2 1 0 0
50 50
GUS activity (nmol min gFW )
GUS activity (nmol min gFW )
8
150
50
100
150
Distance from petiole (mm)
Fig. 5 The spatial distribution of GUS activity in leaf No.8 on 30 d after seeding (left) and the relation between the
80
Leaf Leaf Leaf Leaf
70 60 50
No.5 No.7 No.9 No.11
40 30 20 10 0 15
25
35
45
55
Day after seeding
GUS activity and the distance from its petiole.
Fig. 7 Time course of the GUS activity of leaves No. 5, 7, 9 Figure 6 shows the GUS activity per gFW of each leaf. The
and 11. Bars indicate standard deviations (n=5 on 25, 30 and
GUS activity in the cotyledons (Nos. 1 and 2) was much
34 d; n=3 on 40 d and 50 d).
higher than that in other true leaves. This is probably because the cotyledons have a much longer leaf age. The GUS activity
The maximum GUS activity (Gmax) decreased for higher
decreased at higher leaf positions, and increased as a leaf aged
leaf numbers (younger leaves) (Table 1). The longevity of
(Fig. 6 right). These results indicate that the accumulation of
leaves in the lower positions was greater than that of leaves in
the GUS protein in a leaf is related to its age.
the higher positions. The greater longevity of the leaves in the lower positions probably contributed the higher accumulation
250
-1
25 d 30 d 40 d 50 d
300
of the GUS protein, and the rapid growth of the leaves in the
80
25 30 40 50
-1
GUS activity (nmol min gFW )
GUS activity (nmol min-1 gFW-1)
350
200 150 100 50
70 60 50
d d d d
higher positions diluted the concentration of the GUS activity per unit of fresh weight. Table 2
40
accumulation.
20 10 0
0 0
10
20
Leaf Number
30
Parameters in equation (2) relating to GUS
30
0
10
20
30
Leaf Number
Leaf No.5 Leaf No.7 Leaf No.9 Leaf No.11
G max 71.0 50.3 39.0 22.8
ra 0.24 0.33 0.30 0.54
ta 29.0 36.0 37.9 37.6
Fig. 6 The GUS activity of each leaf. The left graph shows all leaves and the right graph shows the leaves above No.4.
4. GUS accumulation per plant GUS activity per leaf of leaf number i (GLi) can be calculated with equations (1) and (2):
To evaluate the time course of the GUS activity, a logistic equation was employed to describe the GUS activity of a leaf
GLi Gi Fwi
(3)
Then, GUS activity per plant (GP) is expressed by the
number i:
G max i Gi 1 e rai ( tai t )
summation of GL:
(2) -1
Where G = GUS activity (nmol min
-1
gFW ); Gmax =
NL
GP GLi
(4)
i 1
maximum GUS activity (nmol min-1 gFW-1); ra =
where NL is the number of leaves.
accumulation rate; ta = accumulation time constant (d); and t
The GUS activity per leaf from No. 13 to 20 increased
= growth time (d).
dramatically from 40 to 50 d, because FW increased
Figure 7 shows that the time course of the GUS activity of
exponentially and GUS activity per FW also increased (Fig.
leaves No. 5, 7, 9 and 11. The GUS protein of leaf No. 5
8). The GUS activity of Nos. 9 to 12 was still stably
increased during the leaf senescence phase (its color was
accumulating without degradation. Consequently, the GUS
yellow and some parts of it were necrotized). The GUS
activity per plant increased exponentially (Fig. 9).
-1
250
GUS activity per leaf (nmol min leaf )
300
-1
OKAYAMA, OKAMURA, MURASE: Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory
87
and their height could exceed two meters with fewer leaves. Bolting is, therefore, unfavorable for cultivating in a plant 25 d
factory.
30 d 200
On the other hand, a recombinant protein that we require
40 d
may be unstable. Stevens et al. (2000) reported that the
50 d
150
amount of antibody in older tobacco leaves was only half of that in younger leaves. This result indicates that the
100
recombinant protein could not be accumulated efficiently in
50
the old leaves. If the recombinant protein is unstable, the efficient cultivation cycle for unstable recombinant protein
0 0
5
10
15
20
25
30
Leaf Number
production may be shorter than that for stable recombinant protein production.
Fig. 8 Time course of the GUS activity per leaf. Bars indicate standard deviations (n=5 on 25 and 30 d; n=3 on 40 and 50d).
V Summary and Conclusions Transgenic
lettuce
plants
containing
an
introduced
CaMV-35S promoter fused to the GUS gene were grown from seeds in a plant factory for 50 d, and the GUS activity in
GUS activity per plant -1 -1 (nmol min plant )
3500
leaves was measured with time. The results indicate that the
3000
GUS protein in lettuce leaves was accumulated very stably
2500
without degradation. Therefore, the old and large leaves
2000
contained a greater amount of the GUS protein. Consequently,
1500
the GUS protein was accumulated exponentially in the lettuce
1000
plants, while the plants grew exponentially.
500
The exponential increase of the amount of the GUS protein
0
indicated that a longer cultivation period is efficient for stable 15
25
35
45
55
Day after seeding
hand, a recombinant protein that we require may be unstable.
Fig. 9 Time course of the GUS activity per plant. Bars indicate standard deviations (n=5 on 25, 30 and 34 d; n=3 on 40 and 50 d).
Verwoerd et al. (1995) reported that the recombinant protein (phytase) in transgenic tobacco plants was stably accumulated in even senescent leaves, where most of the proteins were degraded. If a recombinant protein that we require can be stably accumulated like these proteins, a longer cultivation period would be effective because lettuce plants grow exponentially. In a conventional plant factory, the weight of a lettuce head at harvest is usually around 150 g. If a longer cultivation cycle can be employed, the final weight of a lettuce head will be much higher, and the diameter and height the
head
will
also
become
If the protein is unstable, the optimum cultivation cycle may be shorter than the conventional cycle. At any rate, the stability of a recombinant protein is the key factor for designing an efficient protein production system.
The GUS protein was much more stable than we expected.
of
recombinant protein production in lettuce plants. On the other
much
greater
than
conventionally grown lettuce. The size of the cultivation bench and the optimum plant density should be reconsidered for an efficient production system (Seginer and Ioslovich, 1999). In addition, if the cultivation period is excessively long, lettuce plants will bolt
A plant factory can provide various environmental conditions for cultivation, including conditions not existing in nature. Environmental control will be also important for controlling the accumulation of the recombinant protein. In stably transformed tobacco, the CaMV-35S promoter was responsive to photoperiod and produced higher expression with a shortened photoperiod than in a longer period (Schnurr and Guerra, 2000). In addition, Joh et al (2005) reported that temperature and lighting conditions affected the GUS expression in transgenic lettuce. Employing promoters which are responsive to environmental stimulation, such as heat, drought, etc., may be useful for enhancing the production efficiency of recombinant proteins in a plant factory. Acknowledgements This research was financially supported by the Japanese Ministry of Economy, Trade and Industry, and conducted as part of the “Development of Fundamental Technologies for
88
Engineering in Agriculture, Environment and Food Vol. 2, No. 3 (2009)
Production of High-value Materials using Transgenic Plants
sensitive to shortened photoperiod in transgenic tobacco. Plant
(2006-2010)” project. We are especially grateful to Dr. Asao
Cell Reports 19: 279-282.
(Nara Prefectural Agriculture Experimental Station), and Dr.
Seginer, I., and I. Ioslovich. 1999. Optimal spacing and cultivation
Sawada and Dr. Matsui (Idemitsu Kosan Co., Ltd) for
intensity
providing the primary transformants, and to Mr. Mizue for his
Agricultural Systems 62: 143-157.
useful advice.
for
an
industrialized
crop
production
system.
Smart, C. M. 1994. Gene expression during leaf senescence. New Phytol. 126: 419-448.
References Ackerly, D. 1999. Self-shading, carbon gain and leaf dynamics: A test of alternative optimality models. Oecologia 119: 300-310. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. Daniell, H., S. J. Streatfield, and K. Wycoff. 2001. Medical molecular farming: Production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science 6: 219-226. Hiatt, A., C. Robert, and K. Bowdish. 1989. Production of antibodies in transgenic plants. nature 342: 76-78. Joh, L. D., T. Wroblewski, N. N. Ewing, and J. S. VanderGheynst. 2005. High-level transient expression of recombinant protein in lettuce. Biotechnology and Bioengineering 91: 861-871. Kapusta, J., A. Modelska, M. Figlerowicz, T. Pniewski, M. Letellier, O. Lisowa, V. Yusibov, H. Koprowski, A. Plucienniczak, and A. B. Legocki. 1999. A plant-derived edible vaccine against hepatitis b virus. FASEB J. 13: 1796-1799. Kim, T.-G., M.-Y. Kim, B.-G. Kim, T.-J. Kang, Y.-S. Kim, Y.-S. Jang, C. J. Arntzen, and M.-S. Yang. 2007. Synthesis and assembly of escherichia coli heat-labile enterotoxin b subunit in transgenic lettuce (lactuca sativa). Protein Expression and Purification 51: 22-27. Kim, Y.-S., B.-G. Kim, T.-G. Kim, T.-J. Kang, and M.-S. Yang. 2006. Expression of a cholera toxin b subunit in transgenic lettuce (lactuca sativa l.) using an agrobacterium-mediated transformation system. Plant Cell, Tissue and Organ Culture 87: 203-210. Li, J., M. Chen, X.-W. Liu, H.-C. Zhang, F. F. Shen, and G. P. Wang. 2007. Transient expression of an active human interferon-beta in lettuce. Scientia Horticulturae 112: 258-265. Morimoto, T., T. Torii, and Y. Hashimoto. 1995. Optimal control of physiological processes of plants in a green plant factory. Control Eng. Practice 3: 505-511. Murase, H. 2002. Precision technologies for protected agriculture. ASAE Paper #023034. Negrouk, V., G. Eisner, H.-i. Lee, K. Han, D. Taylor, and H. C. Wong. 2005. Highly efficient transient expression of functional recombinant antibodies in lettuce. Plant Science 169: 433-438. Okayama, T., K. Okamura, and H. Murase. 2008. A simulation model for heterologous protein production in transgenic lettuce. Environ. Control Biol. 46: 195-202. Schnurr, J. A., and D. J. Guerra. 2000. The camv-35s promoter is
Smith, M. D. 1996. Antibody production in plants. Biotechnology Advances 14: 267-281. Stevens, L. H., G. M. Stoopen, I. J. W. Elbers, J. W. Molthoff, H. A. C. Bakker, A. Lommen, D. Bosch, and J. W. 2000. Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tabacco. Plant Physiology 124: 173-182. Streatfield, S. J., J. M. Jilka, E. E. Hood, D. D. Turner, M. R. Bailey, J. M. Mayor, S. L. Woodard, K. K. Beifuss, M. E. Horn, D. E. Delaney, I. R. Tizard, and J. A. Howard. 2001. Plant-based vaccines: Unique advantages. Vaccine 19: 2742-2748. Sun, H.-J., M.-l. Cui, B. Ma, and H. Ezura. 2006. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Letters 580: 620-626. Torres, A. C., D. J. Cantliffe, B. Laughner, M. Bienick, R. Nagata, M. Ashraf, and R. J. Ferl. 1993. Stable transformation of the lettuce cultivar South Bay from cotyledon explants. Plant Cell, Tissue and Organ Culture 34: 279-285. Verwoerd, T. C., P. A. van Paridon, A. J. van Ooyen, J. W. van Lent, A. Hoekema, and J. Pen. 1995. Stable accumulation of aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol. 109: 1199-1205. (Received : 11. December. 2008, Accepted : 3. March. 2009)
EAEF 2(3) : 83-88, 2009
Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory Tsuyoshi OKAYAMA*1, Kenichi OKAMURA*1, Haruhiko MURASE*2 Abstract Leaf lettuce (Lactuca sativa L. cv. ‘Greenwave’) containing an introduced Cauliflower mosaic virus-35S promoter fused onto the the
-glucuronidase (GUS) gene was grown in a plant factory for 50 days from seeding.
Measurements of the GUS expression level of leaves indicated that the GUS protein that accumulated in the leaves was very stable and did not degrade. Consequently, the GUS expression per plant increased exponentially as the cultivation period became longer. This stability of the recombinant protein indicates that a longer cultivation period would be more efficient than the conventional cultivation period in a plant factory. However, the optimum cultivation period depends on the stability of the recombinant protein which is required. [Keywords] CaMV-35S promoter, -glucuronidase, leaf lettuce, plant factory, transgenic
I
Introduction
Since 1989 when the antibody from mouse was expressed in transgenic tobacco by Hiatt et al. (1989), transgenic plants have been considered promising systems for producing useful protein in bulk amounts at relatively low cost (Smith, 1996; Daniell et al., 2001; Streatfield et al., 2001). If useful proteins for oral administration, such as vaccines, can be produced in edible plants, the purification procedures can be eliminated (Daniell et al., 2001). Lettuce is one of the most popular vegetables in the world, and has already been examined as a host plant for heterologous protein production (Torres et al., 1993; Kapusta et al., 1999; Joh et al., 2005; Negrouk et al., 2005; Kim et al., 2006; Sun et al., 2006; Kim et al., 2007; Li et al., 2007). Lettuce is cultivated commercially in many plant factories in which environmental conditions such as air temperature, relative humidity, light intensity and CO2 concentration are controlled precisely (Morimoto et al., 1995; Murase, 2002). Lettuce has a short cultivation cycle from germination to harvest, and requires relatively low light intensity. In addition, the environment for cultivating lettuce is easy to control, because lettuce is usually cultivated during only its vegetative growth stage. These advantages are important for exploring the relationship between environmental growth conditions and heterologous protein production in a short research period.
When a transgenic lettuce plant is used as a host plant to produce heterologous proteins, most of the proteins will be stored in leaves. Therefore, maximization of total leaf fresh weight seems to be the optimum way to maximize the amount of the heterologous protein. However, the amount of proteins in a leaf decreases because of senescence and environmental stress (e.g. drought, temperature, shading, etc.) (Smart, 1994). Stevens et al. (2000) investigated the relationship among leaf senescence, environmental conditions (temperature and light intensity) and the amount of antibodies (IgG) expressed in tobacco leaves. They reported that the top (young) leaves contained about twice the amount of antibodies as the base (old) leaves. This indicates that the maximization of leaf fresh weight does not equate to the maximization of the amount of heterologous protein in the plant. Smith (1996) noted that the way in which heterologous protein is accumulated in plants needs to be revealed before the technology can be used commercially. Therefore, a new comprehensive criterion is required for maximizing the amount of heterologous protein in plants. Okayama et al. (2008) developed a model for simulating heterologous protein production in lettuce. The model, which focused on the time course of fresh weight and protein concentration of each leaf, indicated the existence of an optimum
cultivation
cycle
and
the
advantages
and
disadvantages of protein production at low and high
*1 JSAM Member, Corresponding author, Osaka Branch, Okayama Lab., NISSHOKU Corporation, 1-2 Naka-ku, Sakai, Osaka 599-8570, Japan; [email protected] *2 JSAM Member, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-1 Naka-ku, Sakai, Osaka 599-8531, Japan
84
Engineering in Agriculture, Environment and Food Vol. 2, No. 3 (2009)
temperatures. In that study, it was assumed that a leaf with a
from lettuce leaves using about 20 mg fresh weight of tissue
higher concentration of total soluble protein (TSP) would be
ground in 250 ul extraction buffer consisting of 50 mM
able to accumulate a higher amount of heterologous protein.
sodium phosphate at pH 7.0, 10 mM EDTA, 0.1% Triton
However, the production and degradation process of
X-100,
heterologous protein appears to be much different from the
2-mercaptoethanol. The homogenate was centrifuged for 5
processes of TSP. Therefore, the heterologous protein should
min at 12,000 rpm at 4 ºC. A soluble extract was used to
be used as a parameter for more practical modelling.
determine GUS activity with 1 mM 4-methylumbelliferyl
0.1%
sodium
lauryl
sarcosyl,
and
10
mM
Although there are many reports about the expression of
- -D-glucuronide as a substrate. The reaction was incubated
heterologous protein in transgenic plants, the cultivation
at 37 ºC for 60 min, and 100 μl aliquot was removed and 2 ml
periods in the most of them were only a few weeks and the
0.2 M Na2CO3 was added to stop the enzyme reaction. The
changes in the amount of heterologous protein per plant over
reaction product 4-methylumbelliferone (MU) was detected
time were not mentioned. Therefore, in this study, transgenic
with a fluorescence spectrophotometer (F-7000, Toshiba Co.,
lettuce plants containing an introduced cauliflower mosaic
Ltd, Japan) at 365 nm excitation/460 nm emission. This GUS
virus (CaMV)-35S promoter fused to the
activity (nmol (MU) min-1) was used for expressing the
-glucuronidase
(GUS) gene were grown in a plant factory which had a
quantitative accumulation of the GUS protein.
conventional cultivation system, and the time course of
TSP concentrations of plant extracts were determined by the
accumulation of heterologous protein in leaves and plants was
dye-biding method of Bradford (1976) with a kit supplied by
investigated to collect information for establishing a practical
Bio-Rad.
heterologous protein production system.
Leaf fresh weight and GUS activity were measured on 25, 30, 34, 40, and 50 d after seeding. Five samples were
II 1.
Materials and Methods
measured on 25, 30 and 34 d and three samples were measured on 40 and 50 d.
Transgenic lettuce
Leaf lettuce (Lactuca sativa L. cv. ‘Greenwave’) was used as the host plant. The binary vector was pBI121 (Clonetech. Inc., USA) containing the neomycine phosphotrasferase II (NPTII) gene, conferring kanamycin resistance, and the GUS
III
Results and Discussion
In this study, leaves were numbered from a cotyledon (Fig. 1).
...
gene containing a functional intron within its coding region under the control of the CaMV-35S promoter. Primary transformants (T0 generation) were self-pollinated and their T1 progenies were self-pollinated to obtain T2 seeds in a closed growth chamber. The seeds were used in the experiment. The percentage of seed germination was consistently over 90%. 2.
Environmental conditions
The transgenic lettuce plants were grown in the plant factory at Osaka Prefecture University, Osaka, Japan. The photo/dark cycle was 14/10 h d-1. Light intensity was approximately 250 μmol m-2 s-1 (photosynthetic photon flux density) and was provided by fluorescent lamps. A half-strength nutrient solution (A-type Otsuka formula, Otsuka Chemical Co., Ltd., Japan) at pH 6.0, electric conductivity of 0.12 S m-1, was supplied to the plants using the deep flow technique. CO2 level was maintained at approximately 1000 μmol mol-1. Air temperature and relative humidity were kept at 23/18 ºC and 50/80% (photo/dark period), respectively. 3. Extraction and GUS assay Quantitative evaluations of GUS expression were carried out following Jefferson et al. (1987). Extracts were prepared
No.8
No.7
No.6
No.5 No.3 No.1
No.4 No.2
Fig. 1 Leaf position and numbering. Nos. 1 and 2 were cotyledons. 1. Relation between GUS activity and TSP The GUS activity and TSP per gram fresh weight (gFW) was measured in 10, 11, 12, and 13 d-old leaves (Fig. 2). The photosynthetic activity becomes the maximum before the leaf expands fully (Hodanova, 1981). Therefore, the protein involved with photosynthesis declines progressively, due to a general decrease in synthesis and an increase in degradation by proteases, and is mobilized to the rest of the plant (Smart, 1994). Our results show that the TSP concentration gradually decreased as a leaf aged. However, the GUS protein kept increasing. This indicates that there is less relation between the respective amounts of TSP and GUS protein and agrees with the high stability of the GUS protein reported by
85
OKAYAMA, OKAMURA, MURASE: Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory Jefferson et al. (1987).
Fwi
Fw maxi Fw max i rgri ( tgri t ) 1 e 1 e rseni (tseni t )
(1)
where Fwmax = maximum leaf fresh weight (g); rgr = 8 GUS activity -1 -1 (nmol min gFW )
7 6 5 4 3 2
GUS activity
1
TSP conc.
0 10
Fig. 2
11 12 Leaf age (d)
growth speed (d-1); tgr = related to onset of growth (d); rsen =
13
Changes in GUS activity and total soluble protein
concentration in a leaf as it ages. Error bars indicate the
senescence (d-1). 14 Leaf Leaf Leaf Leaf
12 10 8
No.5 No.7 No.9 No.11
6 4 2 0 15
standard deviation (n=5). 1.
senescence speed (d-1); and tsen = related to onset of
Leaf fresh weight (g)
10 9 8 7 6 5 4 3 2 1 0
Total soluble concentration -1 (mg gFW )
9
25
35
45
55
Day after seeding
Fig. 4 Time course of fresh weight of leaves No.5 to 11.
Leaf fresh weight
The number of leaves per plant was 8, 10, 18, and 28 on 25,
Bars indicate standard deviations (n=5 on 25, 30 and 34 d; n=3 on 40 and 50 d).
30, 40, and 50 d, respectively (Fig. 3 left). The total leaf fresh weight per plant exponentially increased and more than doubled from 40 d (69.8 ± 11.9 g) to 50 d (190.2 ± 41.6 g)
Leaves in the higher positions grew faster and had higher
(Fig. 3 right). Leaves from No.1 to 4 perished by 40 d, and
fresh leaf weight than leaves in the lower positions (Table 1).
leaves No. 5 and 6 perished by 50 d. The reason of the
When a leaf was shaded by other leaves above it, its fresh
perishing was senescence with aging and shading by higher
weight decreased because of senescence.
leaves (Ackerly, 1999). Table 1 25 d 30 d 40 d 50 d
16 Leaf fresh weight (g)
Total leaf fresh weight per plant (g)
18 14 12 10 8 6 4 2 0 0
5
10
15
20
Leaf Number
25
30
Parameters in equation (1) relating to changes in leaf fresh weight.
250
Leaf No.5 Leaf No.7 Leaf No.9 Leaf No.11
200 150 100
Fw max 0.8 3.9 8.8 9.5
rgr 0.51 0.52 0.69 0.61
tgr 23.6 29.9 33.4 36.2
rsen 0.85 1.12 0.60 -
tsen 40.48 50.32 51.73 -
50 0 25 d
30 d
40 d
50 d
Day after seeding
2. GUS activity The GUS activity was not uniform within the leaves (Fig.5); it increased exponentially from the base to the tip.
Fig. 3 Leaf fresh weight (left) and total leaf fresh weight
This is because the tip developed earlier than the base.
per plant (right), bars indicate standard deviations (n=5 on 25
Therefore, the period for production of GUS protein in the
and 30 d; n=3 on 40 and 50 d).
acral cells was longer than that in the basal cells. In the following GUS activity measurement, the sample was
Figure 4 focuses on the time course of the fresh weight of
extracted from the middle of a leaf, because the GUS activity
leaves No. 5, 7, 9 and 11. Leaf No. 5 perished by 40 d, and the
in the middle part represents approximately the mean value of
color of leaf No. 7 was yellow (almost perished) on 50 d. In
the GUS activity in a whole leaf.
order to evaluate these data objectively, the time course of leaf fresh weight (Fw) (g) of leaf No. i at growth time (t) (d) can be expressed with the following equation including two logistic functions (Okayama et al., 2008). One logistic function describes the growth and the other describes senescence:
86
Engineering in Agriculture, Environment and Food Vol. 2, No. 3 (2009) activity of other leaves was nearly saturated after 40 d.
-1
50
0 00
mm
y = 0.1238e R2 = 0.7187
90 -1
6
-1
5 4
-1
GUS activity (nmol min-1 gFW-1)
mm
100
0.0294x
7
3 2 1 0 0
50 50
GUS activity (nmol min gFW )
GUS activity (nmol min gFW )
8
150
50
100
150
Distance from petiole (mm)
Fig. 5 The spatial distribution of GUS activity in leaf No.8 on 30 d after seeding (left) and the relation between the
80
Leaf Leaf Leaf Leaf
70 60 50
No.5 No.7 No.9 No.11
40 30 20 10 0 15
25
35
45
55
Day after seeding
GUS activity and the distance from its petiole.
Fig. 7 Time course of the GUS activity of leaves No. 5, 7, 9 Figure 6 shows the GUS activity per gFW of each leaf. The
and 11. Bars indicate standard deviations (n=5 on 25, 30 and
GUS activity in the cotyledons (Nos. 1 and 2) was much
34 d; n=3 on 40 d and 50 d).
higher than that in other true leaves. This is probably because the cotyledons have a much longer leaf age. The GUS activity
The maximum GUS activity (Gmax) decreased for higher
decreased at higher leaf positions, and increased as a leaf aged
leaf numbers (younger leaves) (Table 1). The longevity of
(Fig. 6 right). These results indicate that the accumulation of
leaves in the lower positions was greater than that of leaves in
the GUS protein in a leaf is related to its age.
the higher positions. The greater longevity of the leaves in the lower positions probably contributed the higher accumulation
250
-1
25 d 30 d 40 d 50 d
300
of the GUS protein, and the rapid growth of the leaves in the
80
25 30 40 50
-1
GUS activity (nmol min gFW )
GUS activity (nmol min-1 gFW-1)
350
200 150 100 50
70 60 50
d d d d
higher positions diluted the concentration of the GUS activity per unit of fresh weight. Table 2
40
accumulation.
20 10 0
0 0
10
20
Leaf Number
30
Parameters in equation (2) relating to GUS
30
0
10
20
30
Leaf Number
Leaf No.5 Leaf No.7 Leaf No.9 Leaf No.11
G max 71.0 50.3 39.0 22.8
ra 0.24 0.33 0.30 0.54
ta 29.0 36.0 37.9 37.6
Fig. 6 The GUS activity of each leaf. The left graph shows all leaves and the right graph shows the leaves above No.4.
4. GUS accumulation per plant GUS activity per leaf of leaf number i (GLi) can be calculated with equations (1) and (2):
To evaluate the time course of the GUS activity, a logistic equation was employed to describe the GUS activity of a leaf
GLi Gi Fwi
(3)
Then, GUS activity per plant (GP) is expressed by the
number i:
G max i Gi 1 e rai ( tai t )
summation of GL:
(2) -1
Where G = GUS activity (nmol min
-1
gFW ); Gmax =
NL
GP GLi
(4)
i 1
maximum GUS activity (nmol min-1 gFW-1); ra =
where NL is the number of leaves.
accumulation rate; ta = accumulation time constant (d); and t
The GUS activity per leaf from No. 13 to 20 increased
= growth time (d).
dramatically from 40 to 50 d, because FW increased
Figure 7 shows that the time course of the GUS activity of
exponentially and GUS activity per FW also increased (Fig.
leaves No. 5, 7, 9 and 11. The GUS protein of leaf No. 5
8). The GUS activity of Nos. 9 to 12 was still stably
increased during the leaf senescence phase (its color was
accumulating without degradation. Consequently, the GUS
yellow and some parts of it were necrotized). The GUS
activity per plant increased exponentially (Fig. 9).
-1
250
GUS activity per leaf (nmol min leaf )
300
-1
OKAYAMA, OKAMURA, MURASE: Time Course of the Expression of the CaMV35S-GUS Gene in Transgenic Lettuce Plants Grown in a Plant Factory
87
and their height could exceed two meters with fewer leaves. Bolting is, therefore, unfavorable for cultivating in a plant 25 d
factory.
30 d 200
On the other hand, a recombinant protein that we require
40 d
may be unstable. Stevens et al. (2000) reported that the
50 d
150
amount of antibody in older tobacco leaves was only half of that in younger leaves. This result indicates that the
100
recombinant protein could not be accumulated efficiently in
50
the old leaves. If the recombinant protein is unstable, the efficient cultivation cycle for unstable recombinant protein
0 0
5
10
15
20
25
30
Leaf Number
production may be shorter than that for stable recombinant protein production.
Fig. 8 Time course of the GUS activity per leaf. Bars indicate standard deviations (n=5 on 25 and 30 d; n=3 on 40 and 50d).
V Summary and Conclusions Transgenic
lettuce
plants
containing
an
introduced
CaMV-35S promoter fused to the GUS gene were grown from seeds in a plant factory for 50 d, and the GUS activity in
GUS activity per plant -1 -1 (nmol min plant )
3500
leaves was measured with time. The results indicate that the
3000
GUS protein in lettuce leaves was accumulated very stably
2500
without degradation. Therefore, the old and large leaves
2000
contained a greater amount of the GUS protein. Consequently,
1500
the GUS protein was accumulated exponentially in the lettuce
1000
plants, while the plants grew exponentially.
500
The exponential increase of the amount of the GUS protein
0
indicated that a longer cultivation period is efficient for stable 15
25
35
45
55
Day after seeding
hand, a recombinant protein that we require may be unstable.
Fig. 9 Time course of the GUS activity per plant. Bars indicate standard deviations (n=5 on 25, 30 and 34 d; n=3 on 40 and 50 d).
Verwoerd et al. (1995) reported that the recombinant protein (phytase) in transgenic tobacco plants was stably accumulated in even senescent leaves, where most of the proteins were degraded. If a recombinant protein that we require can be stably accumulated like these proteins, a longer cultivation period would be effective because lettuce plants grow exponentially. In a conventional plant factory, the weight of a lettuce head at harvest is usually around 150 g. If a longer cultivation cycle can be employed, the final weight of a lettuce head will be much higher, and the diameter and height the
head
will
also
become
If the protein is unstable, the optimum cultivation cycle may be shorter than the conventional cycle. At any rate, the stability of a recombinant protein is the key factor for designing an efficient protein production system.
The GUS protein was much more stable than we expected.
of
recombinant protein production in lettuce plants. On the other
much
greater
than
conventionally grown lettuce. The size of the cultivation bench and the optimum plant density should be reconsidered for an efficient production system (Seginer and Ioslovich, 1999). In addition, if the cultivation period is excessively long, lettuce plants will bolt
A plant factory can provide various environmental conditions for cultivation, including conditions not existing in nature. Environmental control will be also important for controlling the accumulation of the recombinant protein. In stably transformed tobacco, the CaMV-35S promoter was responsive to photoperiod and produced higher expression with a shortened photoperiod than in a longer period (Schnurr and Guerra, 2000). In addition, Joh et al (2005) reported that temperature and lighting conditions affected the GUS expression in transgenic lettuce. Employing promoters which are responsive to environmental stimulation, such as heat, drought, etc., may be useful for enhancing the production efficiency of recombinant proteins in a plant factory. Acknowledgements This research was financially supported by the Japanese Ministry of Economy, Trade and Industry, and conducted as part of the “Development of Fundamental Technologies for
88
Engineering in Agriculture, Environment and Food Vol. 2, No. 3 (2009)
Production of High-value Materials using Transgenic Plants
sensitive to shortened photoperiod in transgenic tobacco. Plant
(2006-2010)” project. We are especially grateful to Dr. Asao
Cell Reports 19: 279-282.
(Nara Prefectural Agriculture Experimental Station), and Dr.
Seginer, I., and I. Ioslovich. 1999. Optimal spacing and cultivation
Sawada and Dr. Matsui (Idemitsu Kosan Co., Ltd) for
intensity
providing the primary transformants, and to Mr. Mizue for his
Agricultural Systems 62: 143-157.
useful advice.
for
an
industrialized
crop
production
system.
Smart, C. M. 1994. Gene expression during leaf senescence. New Phytol. 126: 419-448.
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