Investigating the Role of
Carotenoids in Root Development
By Grace Scott
Why I am excited about this project...
• The ways which agriculture can help people
• Sustainably feeding the planet
• How we can use science to improve agriculture
• Genetically modified organisms and their benefits
(Benion, 2013)
(Dario, 2015)
Carotenoids
• Natural yellow, orange or red pigments (Olsen and Krinsky, 1995)
• Biosynthethised photosynthetic organisms (Cazzonelli, 2011)
• Important for plant hormone production
• Precursors for Vitamin A (Ye et al, 2000)
(Dentone, 2015)
Vitamin A
• Fat soluble retinoids
• Plant sources: beta-carotene, alpha-carotene or
beta-cryptoxanthin
• Oxidatively cleaved within body to form retinol
(Cazzonelli, 2011) (Olsen and Krinsky, 1995)
• One of the world’s most common micronutrient
deficiencies
(Ye et al, 2000)
(Unicef, 2010)
VAD and Golden Rice
• Rice: cheap, low input and often only source of food
• Eye problems: xerophalmia and keratomalcai
• Compounding factor in 2 million deaths per year (Spady, 2013).
• Golden Rice GMO able to produce β carotene (Ye et al, 2000)
(Unicef, 2010)
(Ye et al, 2000)
• Investigate the below ground effects of carotenoids
• Downstream root hormones hyperexpressed
(strigolactones and abscisic acid)
• Higher root biomass correlates with carotenoid
levelss
• Lateral root branching and adventitious roots increase
Aim:
Hypothesis:
(Cazzonelli, 2011)
Materials
• No golden rice? Work backwards!
• Tomatoes produce high levels of carotenoid trans-
lycopene (Khachik, Carvalho, Bernstein, and Muir, 2002)
• CRTISO mutant does not isomerise carotenoids,
makes tetra-cis-lycopene instead (Eckardt, 2002)
• Essentially a “non carotenoid” prototype
(Cazzonelli, 2011)
(Eckardt, 2002)
Materials
• D15: a chemical inhibitor that acts on the CRTISO
pathway(Van Norman, 2004)
• Aryl-C3N hydroxamic acid analog
• Inhibits enzymes which cleave in the 9,10 position in
carotenoids (Sergent et al, 2009)
• Mimics CRTISO mutation
• $$$$$$$$$$ (Isaacson, 2004)
Experiment 1: Biomass
• Grow in equal conditions in glasshouse
• Collect, prepare and weigh biomass samples(Ostwald and
Ravindranath, 2008)
• Prepare and grow in Phytagel™ agar
• Phenotype roots using Image J (Abdul Muhsin, 2012)
• Compare length and root branching using statistical
analysis
Experiment 2: Root phenotype
Experiment 3: D15 with Phytagel
• Grow with D15 in phytagel
• Chemical inhibitor that blocks the
CRTISO pathway
• Compare length and root
branching using statistical analysis
Results
• 54% higher root biomass/total in
CRTISO after 3 months P<0.05
• 39% higher root biomass/total in
CRTISO after 3 weeks P<0.016
• Unexpected!!
Experiment 1: Biomass
Experiment 2: Root phenotype
• Wildtype has a longer primary root length
by 14.64%, p<0.01
• Wildtype has a higher rate of root
branching by 40%, p<0.01
Experiment 3: D15 with Phytagel
• As the concentration increased, the number of
root branch points decreases in the Wildtype
by 26.56% from the control to the 60mg/L,
P<0.05
• 6.4 lateral roots per plant at 0mg/L of D15
• 1.7 lateral roots per plant at 60mg/L of D15
• Root length differentiation P>0.05
• Null hypothesis
Conclusions
• CRTISO mutant:
1. Shorter root when compared to the wildtype
2. Less lateral root branching
3. Lower overall biomass, but a higher biomass allocation within the roots
• D15 conc. >30micrograms/L causes shortening, less branching and reduced
growth in roots
• ∴ Carotenoid biosynthesis factors in root branching, length and biomass
Implications
• Disfunction of trans-lycopenes = root shortening,
less branching and higher biomass allocation
• Could a hyper production of trans-lycopene
cause the opposite?
• Could golden rice have a better root system than
conventional rice?
(Ye et al, 2000)
(Smith and Read, 2006)
Future Experiments
• HPLC to test levels of 9-cis-β-carotene, 9-cis-
violaxanthin and 9′-cis-neoxanthin within root tissue
• Short roots: inhibition of cell elongation or division?
• Mycorrhizal symbiosis x strigalactones
• Testing on golden rice!
(Boyle et al, 2006)
(Deacon, 1998)
Acknowledgements
• Hawkesbury Institute for the Environment in
partnership with Western Sydney University
• Supervisor: A/Prof. Zhong Hua Chen
• Dr Chris Cazzonelli
• PhD students Yagiz Algoz, Namraj Dhami,
Eric Bren, Rohan Riley, and Michelle Mak
Questions?