Expression of cyanobacterial ictB in higher plants did not enhance photosynthesis. Leonid Asipov

1. 1
Expression of cyanobacterial ictB in higher plants did not enhance photosynthesis.
Leonid Asipov
Abstract
In 2005, an article was published (Lieman-Hurwitz 2005) and contained results of comparison between wt plants and
plants transgenic with ictB, protein which was considered to be involved in carbon transport in cyanobacteria
(Bonfil 1998). The idea was to enhance the photosynthesis of plants by elevation of CO2 concentration near the fixation
site. Elevation of photosynthesis causes elevation in the agricultural yield. The results which were presented showed
that the transgenic plants were better than wt under limiting CO2 conditions and the conclusion was that the protein is
good as an agricultural yield enhancer. However, only partial data was presented and it was not clear from the results,
on which conditions exactly, the transgenic plants had advantage. If natural atmosphere CO2 concentration (around 400
µL L-1 CO2) is not considered "limiting", it is not understandable how the protein will enhance the photosynthesis of
agricultural crops under normal conditions. In the current article, the full data from the same research work is presented
and statistical significance test is calculated in every [CO2] point, comparing wt and transgenic plants. In such way, it is
shown exactly at which [CO2] point the transgenic plants have advantage. The only two points of [CO2] at which the
transgenic plants had significant advantage are 50 and 100 µL L-1, values which are too low for normal plant growth
(Gerhart 2010). Second generation of plants (data which was hidden by the author of the original article) showed no
significant difference relative to the wt plants in all the [CO2] points. Contrary to the original article, our conclusions are
that the transgenic protein did not improve photosynthesis at most of the CO2 points and should not be used as an
agricultural yield enhancer.
Keywords: ictB, photosynthesis enhancement, gas-exchange, transgenic plants

2. 2
Introduction
Presentation of all the data from a research work in important for proper understanding and right conclusions. The
writer of the original article (Lieman-Hurwitz 2005), have hidden most of the data and did not let the readers to
conclude their conclusions based on presented results. Instead, the readers were supposed to believe the author's
conclusions without the ability to argue. A single transgenic plant was presented and the conclusion that transgenic
plants are better only at limiting [CO2] conditions was based on not presented plants. The desire of the readers is to view
all the experimental results and conclude conclusions based on presented scientific proof. Because of that reason, all the
data should be presented, statistical significance tests should be calculated and the conclusions are to be based on
presented data only. Following is the full data presentation of both generations of transgenic plants, including statistical
analysis.
Results
First generation of plants.
In the first generation of transgenic plants, 31 plants were sampled vs 7 wt. All the results are shown in Fig. 1 in a form
of a Ci (intercellular CO2 concentration) vs photosynthesis curve.
Fig. 1 Generation I of plants. Ci vs Photosynthesis curve. Transgenic light-gray, wt black
Large variation is seen in plants photosynthesis. Few transgenic plants are better photosynthetically but most are same
or worse than wt. To quantify the differences between the plants, an average series of wt and the transgenic plants was
calculated (Fig. 2).

3. 3
Fig. 2 Average series of wt and transgenic plants. Ci vs Photosynthesis curve. Transgenic N=31 wt N=7.
Transgenic light-gray, wt black
The wt plants are better at all points except the first three (50, 100 and 200 µL L-1). At 200 µL L-1, there are no visible
differences between wt and the transgenic plants. In the following chart the photosynthetic properties of transgenic and
wt plants have been statistically compared. The T test was calculated for each Ca (ambient CO2 concentration) point
separately.
Fig. 3 Statistical T test of photosynthesis values at every Ca point. p=0.05. Transgenic light-gray, wt dark gray

4. 4
In the first two points (50 and 100 µL L-1) the differences between wt and transgenic plants are significant but
due to the small photosynthesis value (0.35 at maximum), meaningless. The CO2 concentration at these points is
insufficient for normal plants growth (Gerhart 2010) and therefore, the differences in the photosynthesis rate of plants
are not agriculturally practical. At 300, 400, 600 and 800 µL L-1 CO2, wt plants photosynthesise significantly better
than the transgenic plants.
Second Generation of Plants
Despite that in the average series comparison, the wt plants were better at most of the CO2 points,
there were few transgenic plants which were individually better than wt. These plants may differ from the rest
genetically (by the amount of expressed protein for example) and thus there is a possibility that the transgenic protein
gives plants a significant advantage in carbon fixation under all CO2 conditions. To verify this hypothesis many plants
were needed and the best transgenic plants were grown for another generation and sampled in the usual manner.
This part of the experiment was not published or mentioned in the original article (Lieman-Hurwitz 2005).
Fig. 4 Ci vs Photosynthesis, generation II. Transgenic light-gray, wt black
No clear differences are seen between the plants. To quantify the differences between the photosynthesis values,
statistical significance test was calculated at each [CO2] point (Fig. 5).

5. 5
Fig. 5 Statistical significance test of photosynthesis values (II generation). Transgenic N=21 wt N=9. p=0.05.
Transgenic light-gray, wt dark gray
The results suggest that there are no significant differences between the transgenic plants and wt.
The best transgenic plants did not pass their good properties to their progeny.
At the second generation, the transgenic protein does not give plants any advantage, even at the smallest CO2
concentrations. The plants are not different.
Plants growth experiments
The authors' results (Lieman-Hurwitz 2005) suggest that at lower humidity, transgenic plants grow better than wt. Such
results are inconsistent with the gas exchange results, which report significantly better wt photosynthesis at 400 µL L-1
[CO2] (Fig. 3), concentration at which all the plants were grown. All the gas-exchange experiments were conducted at
RH of less than 22%, value which is considered, according to the article, as "low humidity" (Lieman-Hurwitz 2005) .
Since, the photosynthesis of wt plants is higher, the growth is expected to be as well.
Discussion
In the first generation, the transgenic plants were better only at 50 and 100 µL L-1 CO2. These concentrations are too
low for normal plant growth (Gerhart 2010) and therefore application of the protein to agricultural crops, which grow at
normal atmosphere conditions, is not practical. In the second generation of plants, there were no differences at any CO2
concentration. The best transgenic plants did not pass their good properties to their progeny. Therefore, it is possible
that their good properties were not due to their genetics. A measurement inaccuracy (such as uneven watering of plants)
can be proposed. The growth experiments results are not consistent with the gas-exchange. All the plants were measured
in "low humidity", at which transgenic plants were shown to have an advantage according to the plant growth results.

6. 6
The CO2 concentration at which the plants were grown was around 400 (normal atmosphere) and no transgenic
advantage was seen in gas-exchange in such concentration of CO2 (Fig. 3).
The author of the original article (Lieman-Hurwitz 2005) has hidden data and presented false conclusions. The wt plants
photosynthesise better at the normal atmosphere CO2 concentration (Fig. 3). This is the reason why the transgenic
protein is not practical for implementation in agriculture.
Materials and Methods
All measurements were made on LICOR LI6400 Portable Photosynthesis System.
The illumination levels were 500 µE. All the plants were well watered before measurement.
The plants were sampled at low humidity (H2O scrubber was turned on).
The plants were prepared and the measurements were made as described in Lieman-Hurwitz 2005.
Acknowledgements
The study was funded as described in Lieman-Hurwitz 2005.
References
(1)
Lieman-Hurwitz J, Asipov L, Rachmilevitch S, Marcus Y, Kaplan A (2005)
Expression of cyanobacterial ictB in higher plants enhanced photosynthesis and growth.
In: Plant Responses to Air Pollution and Global Change. Springer, Japan, pp 133-139
(2)
Bonfil D J, Ronen-Tarazi M, Sültemeyer D, Lieman-Hurwitz J, Schatz D, Kaplan A (1998)
A putative HCO3- transporter in the cyanobacterium Synechococcus sp. strain PCC 7942.
FEBS Lett 430(3):236-40
(3)
Gerhart L M, Ward J K (2010) Plant responses to low [CO2] of the past.
New Phytol 188: 674–695