First Steps to C4 Alfalfa

Why Alfalfa (Medicago sativa)?● Single largest water user in California● Extremely important cash crop● Great feed for cattle, beef

and dairy● Fixes Nitrogen in soil

o used in crop rotation● Important export to Asia● Currently a C3 plant

C3 Photosynthesis Pros and Cons● Older form of photosynthesis● Found in 85% of plants● Works well in cool, wet conditions with more

CO2

● Uses less energy● Uses more water● Less efficient in hot and dry conditions● Often uses less efficient photorespiration

C4 Photosynthesis Pros and Cons● Newer form of photosynthesis, evolved ~30 million

years ago, independently evolved over 60 times● Very rare● Uses more energy (Carbon must be pumped)● Needs less water● More efficient in hot, dry, conditions● Little to no photorespiration

C3 Mechanism● CO2 enters cell through stomata● Rubisco catches it, forms an

unstable 6 carbon intermediate ● RuBP turns it into 3-PGA (where

C3 comes from) and it is split off into sugars

● Rubisco can catch oxygen, in a wasteful process called photorespiration

C4 Mechanism● Carbon is collected in Mesophyll

cells as a 4 carbon acid● Carbon is turned into

oxaloacetate, then malate● The Carbon then enters the

bundle sheath cells, where CO2 is reformed

● The Calvin Cycle (in C3 photosynthesis) is then used

● Rubisco isn’t exposed to the atmosphere, so little to no photorespiration

Researchable QuestionHow can we alter alfalfa leaves to produce Kranz anatomy?

Medicago truncatula● Close Relative of alfalfa● 8 Chromosomes● Sequenced● Used as a model plant

for comparing genes

● Very common C4 plant● Extremely high yields● Entire genome sequenced● Mechanism of C4

photosynthesis well studied

Zea mays (Maize)

Genes of Interest (All Maize derived transcription factors)● Scarecrow (SCR) ● SHR (Short-Root)● SCR-Like 23● Glk1● G2

The Scarecrow Gene● Helps regulate asymmetric cell development

for Kranz anatomy ● Version found in Medicago● Expressed in Bundle Sheath Cells (BSC)

SHR (Short-Root)● Works with Scarecrow and SCR-Like 23● Controls asymmetric cell development● Expressed in BSC

SCR-Like 23● Controls BS cell specification where it is

expressed● Works with Scarecrow and SHR

Glk1● Expressed in Mesophyll Cells● Believed to regulate Mesophyll and Bundle

Sheath cell differentiation in C4 plants● Also believed to be essential for maize seed

development

G2● Helps control chloroplast development in C4

plants● Bundle Sheath cell specific

Agrobacterium tumefaciens● Common bacterium, used to genetically

modify plants thanks to its Ti plasmid● Genes in between the left and right border

regions are inserted into plantso In the wild, they usually cause

tumourso The tumour causing genes can be

deleted and replaced with more useful genes

o Uses the virulence region to infect plants

● Just one cell needs to be transformed, as plants can be regenerated

Methods Overview1. Engineer Ti Plasmids2. Agrobacterium Mediated Plant

Transformation/Regeneration3. Cross Breeding4. Testing for Results

Engineering Ti Plasmids Part 1● Isolate DNA from maize leaf tissue● Use PCR to amplify the 5 genes of interest

(GOI) using primers designed from known sequences

● Gel purify and clone PCR products

Engineering Ti Plasmids Part 2● Use standard recombinant DNA techniques,

to produce 5 Ti plasmids, each with a separate GOI, along with kanamycin resistance and a promoter o Use ZjPCK for BSC specific genes, use PPCZm1 for

Mesophyll specific genes● Reintroduce Ti plasmids into Agrobacterium

tumefaciens via electroporation

Agrobacterium Mediated Plant Transformation/Regeneration Part 1● Sterilise laminar flow hood, tools● Dip Alfalfa cotyledons in agrobacterium

solution, blot dry, place on B5H medium, leave for 4 days, rinse in water

● Transfer to B5H medium containing kanamycin ● Transfer transformed plants to B5H media with

antibiotics, but not hormones within 3 weeks

Agrobacterium Mediated Plant Transformation/Regeneration Part 2● After 3-4 weeks in B5H w/o growth hormone,

transfer plants to MS Media● Include a control plant, without any

transgenes, and plant in MS Media● Slowly transition plants into a controlled

greenhouse environment

Cross Breeding● Breed together the plants with separate GOI● Use PCR to select plants with

correct transgene● Breed again, until you get a plant

with all the transgenes● Keep controls with no transgenes,

and all combinations of transgenes

Testing for Results: Observing Kranz Anatomy● Cut a small portion of leaf tissue● Prepare microscope slides● View plants● Compare plants

with all transgenesto control plants

Testing for Results: Protein Expression● Perform Western Blot

o Use leaf tissueso Unfortunately, no commercial antibodies are

available for Zea Mays derived SCR, SHR, SCL-23, Glk1, and Glk2

o Custom design an antibody

Expected Results● Absolutely no idea● Nothing similar has ever been done● Hopefully, at least the plant with all the

genes will have rudimentary Kranz anatomy● Not all the genes related to Kranz anatomy

have been discovered yet, so perhaps there are more genes necessary for Kranz anatomy expression

BibliographyGao, Xiaorong, Chaolun Wang, and Hongchang Cui. "Identification of Bundle Sheath Cell Fate Factors Provides New Tools for C3-to-C4 Engineering." Plant Signaling & Behavior 9.6 (2014): n. pag. Web. 29 July 2015.Hanson, Blanie. "Alfalfa." UC Drought Management. University of California, Davis, n.d. Web. 29 July 2015. <http%3A%2F%2Fucmanagedrought.ucdavis.edu%2FAgriculture%2FCrop_Irrigation_Strategies%2FAlfalfa%2F>.Kausch, Albert P., T. Page Owen, Jr., Susan J. Zachwieja, Adam R. Flynn, and Jen Sheen. "Mesophyll-specific, Light and Metabolic Regulation of the C4 PPCZm1 Promoter in Transgenic Maize." Plant Molecular Biology 45.1 (2001): 1-15. Print.Nomura, M. "Differential Expression Pattern of C4 Bundle Sheath Expression Genes in Rice, a C3 Plant." Plant and Cell Physiology 46.5 (2005): 754-61. Web. 29 July 2015.Rossini, Laura, Lizzie Cribb, David J. Martin, and Jane A. Langdale. "The Maize Golden2 Gene Defines a Novel Class of Transcriptional Regulators in Plants." The Plant Cell 13.5 (2001): 1231. Web. 29 July 2015.Slewinski, T. L., A. A. Anderson, C. Zhang, and R. Turgeon. "Scarecrow Plays a Role in Establishing Kranz Anatomy in Maize Leaves." Plant and Cell Physiology 53.12 (2012): 2030-037. Web. 29 July 2015.Slewinski, Thomas L., Alyssa A. Anderson, Simara Price, Jacob R. Withee, Kimberly Gallagher, and Robert Turgeon. "Short-Root1 Plays a Role in the Development of Vascular Tissue and Kranz Anatomy in Maize Leaves." Molecular Plant 7.8 (2014): 1388-392. Web. 29 July 2015.Slewinski, Thomas L. "Using Evolution as a Guide to Engineer Kranz-type C4 Photosynthesis." Frontiers in Plant Science Front. Plant Sci. 4 (2013): n. pag. NCBI. Web. 29 July 2015.Waters, Mark T., and Jane A. Langdale. "The Making of a Chloroplast." Emboj 28.19 (2009): n. pag. 10 Sept. 2009. Web. 29 July 2015. <http://emboj.embopress.org/content/28/19/2861>.

Image Credits (in order of appearance)1. feedyardfoodie.wordpress.com2. farmingamerica.org3. irrri.org (for both animations)4. NCBI Blast5. discoverbiotech.com6. biogeonerd.blogspot.com