1) The study aimed to understand the role of amino acid transporters MtN21-org and MtN21-3 in nitrogen fixation in Medicago truncatula root nodules formed during symbiosis with Sinorhizobium meliloti bacteria. 2) RNAi constructs targeting the transporters were used to transform M. truncatula roots, which were then inoculated with S. meliloti. 3) Preliminary results from acetylene reduction assays showed slightly higher nitrogen fixation in plants transformed with MtN21-org RNAi, though activity levels and sample sizes were low. Further replication was needed.

1. Understanding functions of Amino Acid Transporters MtN21-org and
MtN21-3 involved in Medicago truncatula Nodulation
Sun, Chongzhi; Garcia, Kevin and Ané, Jean-Michel
Department of Bacteriology, University of Wisconsin-Madison
Introduction
Medicago truncatula (Figure 1) is a plant species that forms root nodules with nitrogen fixing
bacteria (e.g., Sinorhizobium meliloti). Plants obtain nitrogen from nitrogen fixing bacteria and
the bacteria receives carbon from plants. In this study we focused specifically on the
mechanism of amino acid transporters of plants, which are responsible for receiving fixed
nitrogen compounds from bacteria. It is known that the amino acid transporters MtN21-org and
MtN21-3 are expressed exclusively in M. truncatula root tissue. We hypothesized that MtN21-
org and MtN21-3 are required for nitrogen fixation to occur within the nodule. Understanding
this mechanism of nitrogen transfer in the nodule will provide further information for how to
engineer nitrogen fixation in other plant species.
Methods
• Seed sterilization: M. truncatula seeds were scarified with sulfuric acid, rinsed with water,
sterilized with a dilute bleach solution, rinsed again, and suspended overnight in sterile
water.
• Seed plating: Sterilized seeds were plated on 1.5% agar with gibberellic acid and incubated
for 3 days at 4°C. The seeds were then incubated at room temperature until they germinated.
• Transformation: Agrobacterium rhizogenes strain MSU440 with RNAi constructs targeting
MtN21-org and MtN21-3, respectively were used to transform M. truncatula (Figure 2).
• Inoculation: Transformed roots were inoculated with Sinorhizobium meliloti (Figure 3).
• Nodule examination and acetylene reduction assay: Transgenic nodules were subjected to–
acetyl reduction assay (ARA) to evaluate levels of nitrogen fixation (Figure 4; Table 1).
Figure 3. Plants inoculated in pots after transformation in first trial.
Transgenic plants were transplanted into pots and acclimated in a plant growth
chamber. The plants were then inoculated with S. meliloti.
Figure 2. Root tissues of M. truncatula transformed with a RNAi construct
targeting MtN21-org (A, B) and with an empty vector control (C, D). These
figures were obtained under dsRed microscopic filter for confirmation of transformed
tissues. Red fluorescence indicates transgenic roots transformed with dsRed and the
RNAi construct. We selected plants with at least partially transformed roots for the
inoculation.
Group name Number in
group
Transformed
nodules
E.V. 1 9
E.V. 2 4
E.V. 3 9
E.V. 4 4
E.V 5 0
E.V. 6 2
E.V. 7 0*
E.V. 8 0
E.V. 9 6
E.V. 10 4
E.V. 11 0
E.V. 12 0
E.V. 13 9
MtN21-org 1 3
MtN21-org 2 3
MtN21-org 3 9
MtN21-org 4 15
MtN21-org 5 1
Figure 4. Representative root
from inoculated RNAi-MtN21-
org plants under brightfield (A)
and dsRed (B) and empty vector
control under brightfield (C) and
dsRed (D).
The plants yielded from inoculation
were reexamined with dsRed
microscopy on whether each nodule
had been transformed. The
untransformed nodules (in darkness
under dsRed) were removed to avoid
the influence of non-treated nodules on
results.
0 0 0.242854411
0.657084317
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Water Ctrl (w/o
nodule)
E.V. RNAi-Mt21-org
NMOLEC2H4/HOUR/PLANT
Nitrogenase Activities of Medicago
truncatula measured by ARA analysis
N mole
C2H4/hour/plant
average
standard
deviation
Water 0
Ctrl (w/o nodule) 0
E.V. 0.242854 0.3821323
RNAi-Mt21-org 0.657084 0.5667609
Results
MtN21-3 group has not provided any useful data since insufficient number of planThe ARA analysis
revealed a major difference of nitrogen fixing capability between the empty vector control and RNAi-Mt-org
(Figure 5). In addition, the large value of standard deviation indicates a random distribution of data with
outliers.
The results, quantitatively, are far below the normal value range (~3-5 mole C2H4/hour/plant). This may be
because the nodules were underdeveloped; therefore, we cannot determine which fixed more nitrogen.
Discussion & Future Research
The experiments gave seemingly negative results because the RNAi-MtN21-org plants displayed stronger
nitrogen fixation activity. However, the activity level is far from the normal range (~3-5 mole C2H4/hour/plant)
as well as the sample size and the number of successful trial the experiment are very limited. This work needs
to be repeated and replicated.
Overly this study does not provide any significant data. That is, we need to proceed the transformation faster,
until we get sufficient samples for the data analysis. In addition, we need to completely clean all containers for
medium to assure the medium components are strictly regulated and the test of RNAi-MtN21-3 group should
also be conducted. Also, seeing that the nodules assessed were not fully developed, the inoculation period
should be extended to about 2-3 weeks so that we can get more mature nodules for normal nitrogen fixation
activity level.
Reference
1. Picture of M. tuncatula (Source: https://www.flickr.com/photos/ajc1/7534380668)
Acknowledgement
This study was supported by the Departments of Bacteriology Agronomy at the University of Wisconsin–
Madison. I would also like to thank the members of the Ané Lab.
A B
C D
A B
C D
Table 1. Plants obtained from
inoculation and the number of nodules
they formed.
* E. V. 7 were removed all nodules and used
as a negative control in ARA analysis.
Figure 1. Picture of Medicago
truncatula.1
Figure 5. Nitrogenase activity levels of M. truncatula plants transformed with RNAi construct
targeting MtN21-org (RNAi-Mt21-org) and with empty vector control (E.V.) with the error bars
referring to standard deviation.