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?