Matthew MerriganJuniorPine Crest School

THE EFFECTS OF ENVIRONMENTAL

STRESSORS ON THE GENETIC MAKEUP OF ROOT

NODULES IN LEGUMES

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

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

LEGUMES

http://www.gardeningknowhow.com/edible/vegetables/vgen/what-are-legumes.htm

http://extension.missouri.edu/publications/DisplayPrinterFriendlyPub.aspx?P=M182

http://academic.reed.edu/biology/Nitrogen/Nfix1(legumes).html

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

RHIZOBIAL RELATIONSHIP

http://www.regional.org.au/au/asa/1987/reviews/p-01.htm

NITRIFICATION

http://faculty.yc.edu/ycfaculty/ags105/week10/soil_organisms/soil_organisms_print.html

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

NOD FACTORS

http://www.waldeneffect.org/blog/A_symbiosis_of_legumes_and_rhizobia/

NOD FACTORS

http://www.utdallas.edu/biology/faculty/research/gonzalez.html

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

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

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

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 extractedContents 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

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

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

A research plan to be conducted would include several components. Testing five different legumes, including chickpeas and others, samples wil l be divided into five different soi l types, collected to display varying soil characteristics. The soil types wil l 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 soi l 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 l ikely that research wil l 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 l ight upon any mutations that may occur within those specific sequences

GENERAL RESEARCH PLAN AND FUTURE RESEARCH

Thank you to Ms. Jennifer Gordinier at Pine Crest School

Also thank you to Dr. Eric von Wettburg at Florida International Univsersity (FIU)

ACKNOWLEDGEMENTS

Faoro, H. "American Society for MicrobiologyAppl ied and Environmental Microbiology." Influence of Soi l Characterist ics on the Diversity of Bacteria in the Southern Brazi l ian Atlantic Forest . 1 Jan. 2010. Web. 30 Jan. 2015. <http://aem.asm.org/content/76/14/4744.ful l>.

Fisher, Robert, Janice Tu, and Sharon Long. "Conserved Nodulation Genes in Rhizobium Mel i lot i and Rhizobium Trifo l i i ." 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.ful l .pdf html>.

Zahran, Hambdi. "American Society for MicrobiologyMicrobiology and Molecular Biology Reviews." Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Condit ions and in an Arid Cl imate . 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 L ibrary of Medicine, 1 Jan. 2010. Web. 30 Jan. 2015. <http://www.ncbi .nlm.nih.gov/pubmed/20022289>.

CITED SOURCES