Are there genes essential for the stimulation of respiration and growth when plants are grown at elevated CO 2 concentrations? Ryan Boyd Andrew Leakey University of Illinois at Urbana-Champaign Department of Plant Biology
Introduction Plants and CO 2 • Elevated atmospheric concentrations of CO 2 stimulate photosynthesis in C 3 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 CO 2 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 CO 2 • How does respiration in plants grown at elevated [CO 2 ] 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 [CO 2 ] is stimulated by 39% compared to soybeans grown under ambient [CO 2 ] (Leakey et al. 2009) • The greater respiratory capacity at elevated [CO 2 ] 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 other 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 [CO 2 ] factors during this screening process
Hypothesis Plants lacking a transcription factor that regulates the increased expression of respiratory genes at elevated [CO 2 ] 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 [CO 2 ] • 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 Arabidopsis thaliana Information Resource)
Materials and Methods Growth Conditions • Plants were grown in environmental growth chambers equipped to elevate [CO 2 ] – Ambient: 400ppm CO 2 – Elevated: 1000ppm CO 2 – 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
Digital Image Analysis Leaf Area Image • 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
Leaf Area Results • The genotype Exposed Leaf Area lacking “Gene 1” and the genotype lacking “Gene 5” gene 1 a 12000 gene 1 e failed to utilize gene 2 a elevated [CO 2 ] to 10000 gene 2 e increase exposed gene 3 a gene 3 e leaf area 8000 2 ) Area (mm gene 4 a • The genotype gene 4 e 6000 lacking “Gene 1” gene 5 a gene 5 e appears to have gene 6 a 4000 larger leaf area gene 6 e throughout the gene 7 a 2000 gene 7 e growing period gene 8 a under ambient gene 8 e 0 conditions gene 9 a 18 22 26 30 34 38 42 46 gene 9 e • The genotype w ild type a Day of Growth lacking “Gene 5” w ild type e showed no difference in leaf area between treatments
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 SPAD readings difference between ambient [CO2] treatments, a trend 35 elevated [CO2] not observed in the wild type 30 • The genotypes lacking 25 “Gene 1” and “Gene SPAD 20 5” were 21.2% and 15 20.4% darker in ambient [CO 2 ] than in 10 elevated [CO 2 ] 5 • This showed that a 0 SPAD meter can be gene 1 gene 2 gene 3 gene 4 gene 5 gene 6 gene 7 gene 8 gene 9 wild type used as a quick phenotyping method in subsequent experiments
Above Ground Mass Results Above Ground Mass ambient [CO2] * 1.4 • Wild type plants elevated [CO2] NS * * NS * showed a 36.9% 1.2 * * stimulation in growth * 1 NS under elevated [CO 2 ] mass (g) 0.8 • The genotypes 0.6 lacking “Gene 1”, “Gene 3” and “Gene 0.4 6” appear to have no significant stimulation 0.2 in above ground biomass when 0 exposed to elevated 1 2 3 4 5 6 7 8 9 e p e e e e e e e e e y n n n n n n n n n t e e e e e e e e e [CO 2 ] d g g g g g g g g g l i w * 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 [CO 2 ] • Wild Type plants grown in elevated [CO 2 ] were larger in area, and had a greater mass than did their ambient grown counterparts
Wild Type and "gene 1" Exposed Leaf Area 10000 Conclusion 8000 gene 1 a area (mm 2 ) gene 1 e 6000 wild type a 4000 wild type e 2000 0 • The three lines lacking “Gene 1”, 18 22 26 30 34 38 42 46 “Gene 3”, and “Gene 5” all failed day of growth to utilize elevated [CO 2 ] in at least one aspect of their growth Wild Type and "Gene 1" SPAD Readings as predicted by the hypothesis 35 30 • The genotype lacking “Gene 1” 25 SPAD 20 ambient [CO2] did not show the typical CO 2 elevated [CO2] 15 treatment effect in area or mass 10 5 when grown under elevated 0 [CO 2 ] conditions gene 1 wild type Wild Type and "Gene 1" • The “Gene 1” line also appeared Above Ground Mass pale, and showed lower SPAD 1.4 1.2 readings in elevated [CO 2 ] than 1 in ambient indicating reduced mass (g) 0.8 ambient [CO2] chlorophyll content elevated [CO2] 0.6 0.4 0.2 0 gene 1 wild type
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 [CO 2 ] • The screening protocol implemented appears to be capable of identifying mutants that respond differently to growth at elevated [CO 2 ] 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 of custom made gas exchange chamber for Arabidopsis leaves • Closed gas exchange system allows for measurement of CO 2 released during respiration over short period of time • Separate compartment allows for temperature control with circulating fluid
Measuring Respiration • 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 CO 2 scrubbers will be able to further sort out CO 2 by plant interactions using the protocol developed by my work in Dr. Leakey’s laboratory
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 [CO 2 ] • 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
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