1. Understanding functions of Amino Acid Transporters
MtN21-org and MtN21-3 involved in Medicago truncatula
Nodulation
Chongzhi Sun
Kevin Garcia, PhD
Jean-Michel Ané, PhD
Department of Bacteriology, University of Wisconsin – Madison

2. Project Objective
 Examine roles of candidate genes in M. truncatula nodulation and nitrogen fixation.
 Transform M. truncatula with empty vector control, RNAi-MtN21-3 and RNAi-
MtN21-org.
 Nodulation analysis

3. Project Rationale
Lodwig et al, 2003

4. 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.
• Inoculation: Transformed roots were inoculated with Sinorhizobium meliloti.
• Nodule examination and acetylene reduction assay: Transgenic nodules were
subjected to– acetyl reduction assay (ARA) to evaluate levels of nitrogen
fixation.

5. Results
0 0 0.242854411
0.657084317
0
0.5
1
1.5
Water Ctrl (w/o
nodule)
E.V. RNAi-Mt21-org
NMOLEC2H4/HOUR/PLANT
Figure 1. 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

6. Results
 MtN21-3 group has not provided any useful data since insufficient
number of plants. The ARA analysis revealed a major difference of
nitrogen fixing capability between the empty vector control and
RNAi-Mt-org (see in Figure 1). 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 treatment
group fixed more nitrogen.

7. Discussion
 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). The sample size were small and only one trial
succeeded. This work needs to be repeated and replicated.
 There is a possibility that nitrogen fixation level will not be
influenced whether the plant receives amino acids produced
bacteriods.

8. Future research
Overly this study does not provide any significant data.
 We need to proceed the transformation faster, until we get sufficient
samples for the data analysis.
 We need to completely clean all containers for medium to assure the
medium components are strictly regulated.
 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.

9. Reference List
 Lodwig E and Poole P. 2003. Metabolism of Rhizobium Bacteriods. Critical Reviews
in Plant Sciences. 22:37-42.