2. Legumes are involved in one of the most well-known and
helpful symbiotic relationships in nature
Rhizobia in the soil aid in the conversion of Atmospheric
Nitrogen (N2) into Ammonia (NH3)
Ammonia can be taken up more easily by the plant, and the
nitrogen can be used more readily
This symbiotic relationship is unique to legumes; it is this
specific interaction that characterizes legumes around the
world
Rhizobia are the only known microorganism living in a
symbiotic relationship with legumes, making this relationship
virtually species-specific
INTRODUCTION
3. Legumes are grown around the world, and provide food across
a variety of environments for a variety of people
In addition, legumes not only provide food, but also provide
green manure, or organic, non-animal waste fertilizer
Legumes are visually distinctive from other plant species by
the presence of a pod, or a bean
Well known legumes include peas, beans, lentils, clovers, and
alfalfa
LEGUMES
5. Rhizobia are symbiotic bacteria found in the root nodules or
nodes of legumes located in the soil
These microscopic organisms aid the legume in the process of
nitrogen-fixation, without which the plants would not be able
to extract the vast quantities of nutrients contained within the
soil
The process of nitrogen fixation involves the conversion of
molecular nitrogen (N2- found in the atmosphere) into
ammonia (NH3- found in the soil).
RHIZOBIA
8. Rhizobia naturally live in the soil, and are attracted by
Vitamin P (or flavonoids- a plant metabolite) that legumes
release into the soil
These flavonoids trigger the bacterium to release nod factors,
which are recognized by the host plant and initiate the
infection of the host plant by the organism. The infection then
leads to cell division, which in turn produces a new root node,
or nodule, constructed as a means of “housing” for the
infectious rhizobia
While much is known about the mutualistic relationship
between the host plant and the infectious organism, very little
is known about the genetic makeup of the root nodes and the
role that environment plays
NOD FACTORS
11. The genes involved in establishing the effective symbiosis in
species of Rhizobium are located on the large plasmid pSym
The gene as a whole is inducible through host plant-secreted
flavonoids. However, in addition to flavonoids, nod expression
also requires the nodD protein, which most likely functions as a
transcriptional activator
After induction, the bacterium secretes a Nod factor. The Nod
factors then induce the steps necessary for nodulation: root hair
deformation, infection, and cortical cell division
Some nod genes, such as nodABCIJ, are highly conserved
amongst Rhizobium species, and share a high degree of
homology among all species of Rhizobia
Because of all of these facts, it serves to reason that an
environmental stressor that results in a mutation of any of the
nod genes would lead to an incorrect or even absent nodulation,
rendering the relationship nonexistent.
NOD GENES
12. Previous studies have demonstrated that varying soil types
do, in fact, have varying effects on the microbial makeup of
any given sample
A general conclusion is that physiochemical characteristics of
soil are based largely on altitude, or depth
To a lesser extent, the altitude affects the Calcium ion to
Magnesium ion ratio. Therefore, this Ca2+/Mg2+ ratio can
play a role in the microbial diversity at any given altitude
Other factors in any given sample of soil play significant roles
in determining the efficacy of the symbiotic relationship. Such
conditions include: salt stress, drought stress, acidity,
alkalinity, nutrient deficiency, fertilizers, heavy metal
concentration, and pesticides
ENVIRONMENTAL STRESSORS
13. The purpose of this experiment is to examine the effects of
various soil qualities (imitating various environmental
stressors) on the genetic makeup of rhizobia of a given
legume
PURPOSE
14. Five different legume species (the common bean, chickpea,
medicago black medic, medicago polymorpha, and the pigeon
pea) will be grown in various soil conditions and various soil
qualities
Nodes will be harvested and contents extracted
Contents will be cultured on a petri dish containing Mannitol
Yeast Agar for approximately five days (until ample growth
has appeared)
DNA will be extracted from the rhizobia utilizing a Qiagen
“Mini-Prep” kit
Extracted DNA will then go through PCR, and sequencing
analyses will be run
MATERIALS AND METHODS
15. Likely results would include a greater rhizobial diversity in soil
samples that were either not autoclaved or from a more
fertile region
This study is intended to elucidate the genetic makeup of the
rhizobia of specific species of legumes, as well as to
determine the specific effects of soil quality on the genomes
of rhizobial nodes
Therefore, it is likely that less fertile soil will demonstrate
decreased genetic diversity
RESULTS
16. It stands to reason that environmental stressors may cause a
mutation in any number of the nod gene family. Because of
the highly conserved nature of the nod gene, a mutation in
this family would implicate the stressors as resulting in a
significant mutation in the root nodule, or a change in the
rhizobia present within the host plant’s nodule
The relationship between environmental stressors and
differences in microbial diversity has already been elucidated,
and yet research has not been conducted with regard to the
environmental stressors and differences in rhizobial
communities
DISCUSSION
17. A research plan to be conducted would include several components.
Testing five different legumes, including chickpeas and others, samples
will be divided into five different soil types, collected to display varying
soil characteristics. The soil types will be either autoclaved or
unautoclaved, to reflect the presence or absence of any nutrients or
other microorganisms present. After waiting for a growth period, rood
nodules would be harvested and the rhizobia present would be
sequenced. The sequences of the autoclaved soil from any one sample
to the natural soil from the same location would be compared as to
demonstrate the effects of “stressors”. This experiment could
strengthen the claims that environments significantly contribute to
microbial diversity in any given area. It is likely that research will
support the idea that microbes are dependent on environmental
factors.
Depending on the sequence analysis of this step, a future research
direction would include the sequencing of the specific Nod factors,
which would shine light upon any mutations that may occur within
those specific sequences
GENERAL RESEARCH PLAN AND FUTURE
RESEARCH
18. Thank you to Ms. Jennifer Gordinier at Pine Crest School
Also thank you to Dr. Eric von Wettburg at Florida
International Univsersity (FIU)
ACKNOWLEDGEMENTS
19. Faoro, H. "American Society for MicrobiologyApplied and Environmental
Microbiology." Influence of Soil Characteristics on the Diversity of Bacteria
in the Southern Brazilian Atlantic Forest. 1 Jan. 2010. Web. 30 Jan. 2015.
<http://aem.asm.org/content/76/14/4744.full>.
Fisher, Robert, Janice Tu, and Sharon Long. "Conserved Nodulation Genes
in Rhizobium Meliloti and Rhizobium Trifolii." Applied and Environmental
Microbiology. U.S. National Library of Medicine, 1 Jan. 1984. Web. 30 Jan.
2015. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC241742/?page=1>.
Hirsch, Ann. "What Makes the Rhizobia-Legume Symbiosis So Special?1."
Plant Physiology. 1 Jan. 2001. Web. 28 Jan. 2015.
<http:/www.plantphysiol.org/content/127/4/1484.full.pdf html>.
Zahran, Hambdi. "American Society for MicrobiologyMicrobiology and
Molecular Biology Reviews." Rhizobium-Legume Symbiosis and Nitrogen
Fixation under Severe Conditions and in an Arid Climate. 1 Jan. 1999. Web.
30 Jan. 2015. <http://mmbr.asm.org/content/63/4/968.abstract>
Von Wettberg, Eric. "Adapting Genomics to Study the Evolution and Ecology
of Agricultural Systems." National Center for Biotechnology Information.
U.S. National Library of Medicine, 1 Jan. 2010. Web. 30 Jan. 2015.
<http://www.ncbi.nlm.nih.gov/pubmed/20022289>.
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