Are there genes essential for the stimulation of respiration and - - PowerPoint PPT Presentation

Ryan Boyd Andrew Leakey

University of Illinois at Urbana-Champaign Department of Plant Biology

Are there genes essential for the stimulation

• f respiration and growth when plants are

grown at elevated CO2 concentrations?

Introduction

Plants and CO2

• Elevated atmospheric concentrations of CO2 stimulate

photosynthesis in C3 plants

• Stimulation of carbon gain will not benefit productivity or

fecundity unless the plant can utilize the increased sugars produced by the increased rate of photosynthesis through the process of respiration (Ainsworth, Rogers & Leakey 2007)

• The release of CO2 as a result of respiration in plants is a major

component of the global carbon cycle (Canadell et al. 2007)

• A better understanding of carbon cycling is needed if a thorough

understanding of climate change and successful adaptive and mitigative approaches are to be implemented

Introduction

Plants and CO2

• How does respiration in plants grown at elevated [CO2] utilize the

increased sugar production?

• Studies at the SoyFACE (Soybean Free Air Concentration

Enrichment) field-research facility revealed that nighttime, foliar respiration of soybeans grown under elevated [CO2] is stimulated by 39% compared to soybeans grown under ambient [CO2] (Leakey et al. 2009)

• The greater respiratory capacity at elevated [CO2] was associated

with greater expression of 627 genes, including those encoding components of the respiratory pathway and 25 genes encoding transcription factors (Leakey et al. 2009)

Introduction

Transcription Factors

• Transcription factors regulate the transcription of
• ther genes
• Individual transcription factors can effect many

important developmental and metabolic processes

• We chose to work with the 25 transcription whose

transcription was altered by elevated [CO2] factors during this screening process

Hypothesis

Plants lacking a transcription factor that regulates the increased expression of respiratory genes at elevated [CO2] will be unable to generate additional energy and utilize additional sugar in order to enhance growth.

The Approach

Reverse Genetics

• Reverse genetic screening allows for a preliminary

look at a gene’s function without in depth research and experimentation

• Using mutants deficient in specific genes we have the

ability to screen for important phenotypes under elevated [CO2]

• This allows us to ask general questions such as: How

does the absence of gene function influence biomass?

The Approach

Using a Model Genetic Organism

• Arabidopsis thaliana is a

model genetic plant

• Arabidopsis is easily

mutated, has a short generation time, is easily grown in a laboratory setting, and has its entire genome sequenced

• Mutants of Arabidopsis

are available from TAIR (The Arabidopsis Information Resource)

Arabidopsis thaliana

Materials and Methods

Growth Conditions

• Plants were grown in

environmental growth chambers equipped to elevate [CO2]

– Ambient: 400ppm CO2 – Elevated: 1000ppm CO2 – Day Length: 10 hours – Light: 250 µmol m-2 s-1 – Humidity: 70% – Temperature: 21°C Day 18°C Night

Environmental Growth Chamber

Measurements

• Measurements taken every 1-3 days

– Digital Pictures – Leaf Area – Leaf Count

• SPAD measurements taken at multiple points

during growth period

• Above ground biomass was collected at the

end of the growing period

• During the first round of screening approximately 3,080

pictures were taken over the course of the growing period

• Digital pictures allows for visual comparisons
• Using Image J software exposed leaf area calculations can be

made

• The program relates the number of pixels to a known size to

calculate area

Digital Image Analysis

Leaf Area Image

Leaf Area Results

• The genotype

lacking “Gene 1” and the genotype lacking “Gene 5” failed to utilize elevated [CO2] to increase exposed leaf area

• The genotype

lacking “Gene 1” appears to have larger leaf area throughout the growing period under ambient conditions

• The genotype

lacking “Gene 5” showed no difference in leaf area between treatments

Exposed Leaf Area

2000 4000 6000 8000 10000 12000 18 22 26 30 34 38 42 46 Day of Growth Area (mm

2)

gene 1 a gene 1 e gene 2 a gene 2 e gene 3 a gene 3 e gene 4 a gene 4 e gene 5 a gene 5 e gene 6 a gene 6 e gene 7 a gene 7 e gene 8 a gene 8 e gene 9 a gene 9 e w ild type a w ild type e

Chlorophyll Approximations

• A SPAD meter is a handheld chlorophyll

meter that can approximate chlorophyll content by calculating the ratio of transmittance through a leaf at red and infra-red wavelengths.

SPAD Results

• The genotypes lacking

“Gene 1” and “Gene 5” show a clear difference between treatments, a trend not observed in the wild type

• The genotypes lacking

“Gene 1” and “Gene 5” were 21.2% and 20.4% darker in ambient [CO2] than in elevated [CO2]

• This showed that a

SPAD meter can be used as a quick phenotyping method in subsequent experiments

SPAD readings

5 10 15 20 25 30 35 gene 1 gene 2 gene 3 gene 4 gene 5 gene 6 gene 7 gene 8 gene 9 wild type SPAD ambient [CO2] elevated [CO2]

Above Ground Mass

0.2 0.4 0.6 0.8 1 1.2 1.4 g e n e 1 g e n e 2 g e n e 3 g e n e 4 g e n e 5 g e n e 6 g e n e 7 g e n e 8 g e n e 9 w i l d t y p e mass (g) ambient [CO2] elevated [CO2]

Above Ground Mass

Results

• Wild type plants

showed a 36.9% stimulation in growth under elevated [CO2]

• The genotypes

lacking “Gene 1”, “Gene 3” and “Gene 6” appear to have no significant stimulation in above ground biomass when exposed to elevated [CO2]

NS NS NS

* Indicates significant difference between treatment within genotype (P<0.005) NS Indicates non-significant differences between treatment within genotype (P>0.05)

* * * * * * *

Results

• As expected the Wild Type plants

showed a stimulation of growth at elevated [CO2]

• Wild Type plants grown in elevated

[CO2] were larger in area, and had a greater mass than did their ambient grown counterparts

Conclusion

• The three lines lacking “Gene 1”,

“Gene 3”, and “Gene 5” all failed to utilize elevated [CO2] in at least one aspect of their growth as predicted by the hypothesis

• The genotype lacking “Gene 1”

did not show the typical CO2 treatment effect in area or mass when grown under elevated [CO2] conditions

• The “Gene 1” line also appeared

pale, and showed lower SPAD readings in elevated [CO2] than in ambient indicating reduced chlorophyll content

Wild Type and "Gene 1" Above Ground Mass

0.2 0.4 0.6 0.8 1 1.2 1.4 gene 1 wild type mass (g) ambient [CO2] elevated [CO2]

Wild Type and "Gene 1" SPAD Readings

5 10 15 20 25 30 35 gene 1 wild type SPAD ambient [CO2] elevated [CO2]

Wild Type and "gene 1" Exposed Leaf Area

2000 4000 6000 8000 10000 18 22 26 30 34 38 42 46 day of growth area (mm 2) gene 1 a gene 1 e wild type a wild type e

Conclusion

• My experiment revealed that “Gene 1” is a

good candidate for being key to controlling the stimulation of metabolism and growth in plants grown at elevated [CO2]

• The screening protocol implemented appears

to be capable of identifying mutants that respond differently to growth at elevated [CO2] than do wild type plants

Follow Ups

• After screening for mutant lines of interest, gas

exchange systems can be used to both measure photosynthetic and respiratory rates

• Coupling gas exchange measurements with

carbohydrate, RNA, carbon/nitrogen ratio, chlorophyll content, and DNA samples can help to characterize metabolic and physiological differences between treatments

Measuring Respiration

• I assisted in the design
• f custom made gas

exchange chamber for Arabidopsis leaves

• Closed gas exchange

system allows for measurement of CO2 released during respiration over short period of time

• Separate compartment

allows for temperature control with circulating fluid

• Experiments aimed at measuring respiration

have not yielded results consistent with previous experiments due to problems experienced with growth chambers

• Future experiments utilizing the newly

installed CO2 scrubbers will be able to further sort out CO2 by plant interactions using the protocol developed by my work in Dr. Leakey’s laboratory

Measuring Respiration

Summary of my work

• I successfully developed a protocol for

screening mutant lines of Arabidopsis including growing, propagating, and phenotyping

• I identified three transcription factors that may

regulate the stimulation of respiration when grown at elevated [CO2]

• I helped to develop a custom respiration

chamber and protocol for measuring respiration of attached leaves

Acknowledgements

• Dr. Andrew Leakey

Cody Markelz Reid Strellner Kevin Dommer Institute for Genomic Biology Global Change Education Program

References

• JG Canadell, C Le Quere , MR Raupach, CB Field, ET Buitenhuis, P

Ciais, TJ Conway, NP Gillett, RA Houghton & G Marland (2007) Contributions to accelerating atmospheric CO2 growth from economic activity, carbon intensity, and efficiency of natural sinks. Proceedings of the National Academy of Sciences 104: 18866-18870.

• EA Ainsworth, A Rogers, ADB Leakey (2008) Targets for crop

biotechnology in a future high-CO2 and high-O3 world. Plant Physiology 147:13-19.

• ADB Leakey, F Xu, K Gillespie, JM McGrath, EA Ainsworth & DR Ort.

(2009) Genomic basis for stimulated respiratory carbon loss to the atmosphere by plants growing under elevated CO2. Proceedings of the National Academy of Sciences. 106: 3597-3602.

• KL Griffin, OR Anderson, MD Gastrich, JD Lewis, G Lin, W Schuster, JR

Seeman, DT Tissue, MH Turnbull & D Whitehead (2001) Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure. Proceedings of the National Academy of Sciences 98: 2473-2478.