Opportunities and Challenges for Canola in the Next 25 Years

– Carbon and Nitrogen Fixation and Science “Fictation”

Murray Hartman Provincial Oilseed Specialist Alberta Agriculture and Forestry

A Lot of Change Over My Career

Canola Wheat Canola Wheat Canola Wheat

Next 25 Years as Exciting?

Opportunities

• C fixation (photosynthesis) • N fixation in Brassicas

Challenges

• Public perception of risks / benefits for agricultural technology

• Science “fictation” in social and mass media

Source: Global rapeseed yield from FAOSTAT

Measuring Response of Brassica Oilseeds to Increased CO2 Concentration e[CO2]

• Range of methods – Growth chamber – Open top chamber in field – Free air concentration enrichment (FACE) in field

• Range of [CO2] increases and duration • Range of plant parts (roots, leaf to whole

plant)

Figure 1 from Messerli et al. 2015 Agron. J. 107 (3): 916-920

SoyFACE (Illinois)

C3 Crops Respond to e[CO2]

• Most terrestial plants and agricultural crops are C3 where RuBisCO is in contact with air via intercellular space and stomatal pores – RuBisCO can react with CO2 or O2

• O2 leads to photorespiration and reduced photosynthetic efficiency

– e[CO2] helps carboxylation vs oxygenation and thus C3 plants should respond more than C4

Recent meta-analysis of 18 OTC and 11 FACE studies for C3 crops

(Bishop et al., Food and Energy Security 2014, 3:33-45)

• Only 1 site for Brassica oilseed (Germany FACE)

• Separated OTC (average e[CO2] 691 ppm) from FACE (average e[CO2] 560 ppm)

Food and Energy Security, Figures 1 and 2 Volume 3, Issue 1, pages 33-45, 29 MAY 2014 DOI: 10.1002/fes3.44 http://onlinelibrary.wiley.com/doi/10.1002/fes3.44/full#fes344-fig-0001

FACE OTC

FACE Germany • Various published papers Högy, Franzaring,

Fangmeir – Agriculture, Ecosystems and Environment 2010 139:239-244

• Mini-FACE (2m diameter) 2007 at Stuttgart-Hohenheim using spring B. napus, irrigated

• 500 ppm target for e[CO2] day and night – Aboveground biomass ↑ 21%, seed yield ↑ 18% – Seeds per m2 ↑17%, 1000K seed weight unchanged

• ↓seed S 6%, Zn 6%, B 4%, seed [protein] 4.6% although protein yield per m2 ↑14%

Additional Brassica FACE / OTC

• Ruhil et al., (Protoplasma 2014: 1-12) – B. juncea FACE rings New Delhi, India 2010-13

• e[CO2] of 585 ppm – “No nutrient or water stress”

• Rabha and Uprety (Photosynthetica 1998 35:597-602) – B. juncea, OTC 2.6m diameter, New Delhi

• e[CO2] of 600 ppm, daytime only – Two moisture treatments reported

• no moisture stress • Stress at flowering when irrigation was withheld 8d from

first flower

Effect of e[CO2] Compared to Ambient on Various Plant Parameters

FACE juncea OTC juncea OTC juncea dry at flower

Upper leaf photosynthetic rate ↑48% ↑29% ↑48%

Leaf transpiration ↓39% ↓25% ↓50%

Leaf area ↑41% ↑78% ↑75%

Above ground dry matter per plant ↑101% ↑39% ↑70%

Root dry matter - ↑28% ↑53%

Seed yield per plant ↑ 24% ↑ 21% ↑88%

1000K seed weight ↑37% ↑8% ↑39%

Seed number per plant ? ↓ ? ↑ ? ↑

Opportunity for Breeding to Capture More Benefit with e[CO2]

• With rising population and income we will need more food than stimulated by e[CO2] – FAO projects global ag production in 2050 will need to

be 60% higher than 2005 (2012 revision) • Brassica germplasm differs in response

– B. campestris / nigra / carinata / juncea in OTC (Mishra et al. 1999 J. Agronomy & Crop Science 182:223-229)

– 6 old and new winter B. napus varieties (Johannessen et al. 2002 Euphytica 128:75-86)

– 4 diverse varieties of spring B. napus (Frenck et al., 2011 Europ. J. Agronomy 35:127-134)

e[CO2] Germplasm Selection Conventional Breeding

• Challenges – Limited plot space in FACE or OTC will limit number of

lines to be screened – Need to screen several Brassica species – No clear consensus on selection criteria

• Without specific morphological, biochemical or physiological traits to select for, then e[CO2] is needed throughout breeding selections

• Four year study of 18 soy genotypes in SoyFACE showed genetic variation for yield under e[CO2] that was consistent enough to be heritable – There wasn’t a photosynthetic basis for this variation

• Bishop et al. 2014, Plant, Cell & Envir.

Beyond Conventional Breeding • Knowledge of 100+ photosynthesis steps has

greatly increased thanks to modern technology • Chloroplast genetic transformation possible for

some crops including B. napus (Schneider et al., 2015, Transgenic Res 24:267-277)

• To achieve the greatest increase in photosynthesis and yield under e[CO2] – Use all tools (FACE, genetic, molecular, imaging,

computational tech) – multi-disciplinary, multi-agency, global collaboration – AND EXPENSIVE

• http://ripe.illinois.edu/

• 5 year project at U. of Illinois 2012-17 • Focus on rice, legumes and cassava for small farmers in developing world

– $25 million grant from Bill & Melinda Gates Foundation – Supercomputer simulations, tobacco test crop for molecular changes

• Multi-institutional in addition to U of Illinois – Australian National University – Rothamsted Research – University of Essex – University of California at Berkeley – Chinese Academy of Sciences-Max Planck Institute – Louisiana State University

• Part of large program at U of I, other related projects – SoyFACE – ARPA-E-PETRO $6.8 million 4 year project with sugarcane, sorghum where one

goal is to modify crop canopy to better capture sunlight – Blue Waters National Center for Supercomputing Applications

Targets in Photosynthesis

• A few examples of many good reviews – Long et al., 2015, Cell 161 (1):56-66 – Singh et al., 2014, Plant Biotech. J. 12(9): 1217-1230 – Sun et al., 2009, Plant science 177 (6): 511-522

• Both light dependent and independent components of photosynthesis – Also sink effects to reduce feedback inhibition – I’ll highlight only a few with Brassica / Arabidopsis

Targets: Light Dependent Reactions • Leaf orientation

– More vertical upper leaves to allow more light to bottom leaves

– Brassica oilseed leaves senesce during pod fill and pods become significant photosynthetic source

• Recent work with Arabidopsis shows inflorescence important as source and sink and was major contributor to whole plant net photosynthesis and C gain

• Leonardos et al., 2014 Environ. and Exp. Bot. 107: 84-97

• Lighter upper leaf color to unshade lower leaves • ARPA-E-PETRO $7m camelina project at Danforth Center, St.

Louis

• Increase electron transfer rate in photosystems to generate ATP and NADPH faster – (Arabidopsis, Chida et al., 2007, Plant Cell Phsyiol. 48: 948-957) – Cold acclimation transcription factors

overexpressed in B. napus (Dahal et al., 2012 Planta 236:1639-1352; Savitch et al., 2005, Plant Cell Physiol. 46(9):1525-39)

Figure 1 from Singh et al., 2014

RuBisCO • Usually identified as the key bottleneck in spite of

being the most abundant protein in plants – specificity for CO2 vs O2 especially at higher

temperature – One of the slowest enzyme reactions

• Substitute or improve RuBisCO – Natural diversity in C3 plants, algae – Plastome modification / editing

• Arabidopsis RuBisCO large subunits expressed in tobacco plastome needed accompanying chaperone protein to achieve 2X increase in photosynthesis and plant growth

– Whitney et al., 2015, PNAS 112(11): 2564-2569 – Cold acclimation transcription factors overexpressed

in B. napus increased RuBisCO content (Savitch et al., 2005, Plant Cell Physiol. 46(9):1525-39)

RuBisCO Activase (RCA)

RuBisCO activase helps RuBisCO function but is sensitive to higher temperatures • Transgenic Arabidopsis plants with tolerant

RCA for heat, ADP / redox improved photosynthesis, biomass, seed yield

• Carmo-Silva and Salvucci, 2013, Plant Physiol. 161:1645-1655

• Kumar et al., 2009, Photosynth. Res. 100:143-153

Faster Calvin Cycle Enzymes and RuBP Regeneration

• under e[CO2] specificity improves but then efficiency affected by RuBP regeneration – Many possible enzyme targets

• Tobacco overexpressing Arabidopsis SBPase had increased photosynthetic rates under FACE

– Rosenthal et al., 2011, BMC Plant Biol. 11:123

• Arabidopsis overexpressing aldolase in chloroplasts increased photosynthesis, biomass and seed yield

– Hatano-Iwaski and Ogawa, 2012, Int. J. Plant Dev. Biol. 6(1):1-8

• Cold acclimation transcription factors

Photorespiration • If RuBP is oxygenated, the C2 compound is

metabolized through complex pathways in mitochondria and peroxisome to regenerate PGA for Calvin cycle, CO2 and NH3 – Large penalty on photosynthetic efficiency

• But participates in NO3 assimilation (Bloom, 2015, Photosynth. Res. 123 (2):117-128)

– Several alternative metabolic pathways from bacteria with better efficiency

• Transgenic Arabidopsis with 3 enzyme step pathway in chloroplast more energy efficient and increased photosynthesis and growth (Kebeish et al., 2007, Nature biotechnology, 25(5), 593-599)

Sink Targets • Increase sink size / rate of CHO export or

feedback inhibition occurs – Possibilities are not well explored

• increased respiratory flux of C into TCA cycle (pyruvate to Acetyl-CoA) with transgenic Arabidopsis using antisense mitochondrial enzyme led to larger inflorescence under e[CO2] (Dahal et al., 2014, Can. J. Plant Sci. 94 (6):1075-1083)

• Other nutrients can’t be limiting – Will soil test calibrations need to be changed?

Nitrogen • The most common nutrient applied, highest cost per acre • Does have adverse environmental effects in some

situations • Improve Brassica N uptake and utilization efficiency

– Approaches already in progress • Hybrids have better scavenging ability (uptake efficiency) • Screening for higher NUE is difficult since it is complex trait • Novel NUE traits have been tested in field trials, still no regulatory

decisions (for example Arcadia Biosciences) • N fertilizer additives (ESN, Agrotain, etc.) • Precision farming

– Field testing for possible synergism e[CO2] / modified photosynthesis

Can We Teach Brassica to Fish? (make its own N fertilizer)

Several methods where Brassica oilseeds could biologically fix atmospheric nitrogen

– Other than crop rotation with legumes • 1. Inoculation of Brassica seeds with free-living /

associative N-fixers – Several genera of naturally existing N fixers – also growth promoting mechanisms and disease

suppression – Competition in rhizosphere, benefit for weeds?

Foreign vs Local Strains?

• Regulatory benefit for using country resident microbes?

• 4% of rhizosphere bacteria in 3 Saskatchewan field isolates from B. napus could fix N (Germida 1998 CARP project final report) – Growth was increased in growth chambers and one

field study – Bacillus polymyxa strain RSN17 should be pursued – Antagonism from seed treatment fungicides? – Are there differences in Brassica germplasm for

favouring rhizobacteria?

Engineer the rhizosphere

• Engineer N fixation into common rhizobacteria – Pseudomonas protegens modified to fix N produced

NH4 in media and improved growth of Arabidopsis and other crops

• Setten et al., 2013 PLOS ONE, 8(5):e63666 – Regulatory hurdles?

• Engineer root exudates to favour certain species – “lock and key” so that only modified crop can sustain

growth of engineered N fixer • Transgenic Arabidopsis exuded opine carbon and shaped

bacterial colonization in rhizosphere – Mondy et al., 2014, Molecular Ecol. 23:4846-4861

2. Endophytic N fixation N fixation in Brassicas using endophytes • Gluconacetobacter diazotrophicus from sugar

cane – 20-60% of N uptake supplied by this endophyte to

sugar cane – Has been found to colonize roots in B. napus and

other crops in lab conditions (Cocking et al, In Vitro Cell. Dev. Plant, 2006)

• Has patented and licensed this technology to Azotic Technologies, called N-Fix, who are working on field trials for efficacy data

3. Symbiotic N fixation • Develop legume like N fixing nodules for Brassicas

– Can Brassicas find a major funder like Bill and Melinda Gates Foundation who has granted $10M to John Innes Center in UK and several US institutions for N fixation in cereals (maize focus)

– Lock and key system • Nodules have been induced in Brassica napus with Rhizobia

or Bradyrhizobia or other agents – Al-Mallah et al, J. Exp. Bot. 1990; Isobe et al. Soil Sci. Plant Nutr.

1995; Trinick and Hadobas, Plant and Soil 1995; Kovalskaya et al. Microbiol. 2001.

– Is this what hybridization nodules are? – The Kovalskaya paper reported nitrogen-fixing ability of plants

inoculated with mix of bacterial free-living N-fixers and 2,4-D induced nodules

Biotech Tools May Provide Solutions But Will the Public Accept Them?

• Technology greatly affects agriculture in both machinery and biological aspects

• Plant genetic engineering is negatively viewed by the public • Numerous public opinion survey

examples • Strong activist organizations • Results in excessive regulation

Technological advances

impact many aspects of

our lives, and are generally

quickly adopted

Pew Research Center, Jan. 2015 Public and Scientists’ View on Science and Society

• Paired surveys in USA with about 2000 citizens and 3700 scientists (American Association for the Advancement of Science, AAAS) – Wide difference in opinion regarding genetically

modified foods – www.pewresearch.org

Is GM Technology Safe??? My Spin…

• Leading cause of death for young people 15-29

• For every one fatality, 20 people suffer injury

• I am assuming 11.5% GM global market share

Based on 2013 World Health Organization information, I

estimate about 143,000 men, women and children die

from GM tech each year

And GM Technology Kills Wildlife Too

• Canadian statistics: about 7,600 large wildlife deaths in 2003

WHO, Global status report on road safety 2013: supporting a decade of action. ** I am not implying GM vehicles are less safe than other makes

• Carcinogens! – Known: diesel engine exhaust, alcohol, mineral oil – Probable: gasoline engine exhaust, asphalt (to

paving workers)

• Kills the agro-ecosystem! – 12 M ha of roads worldwide (Jacobson and Ten Hoeve,

2012, J. Climate 25:1028-1044)

• Slaughters wild pollinators and beneficial insects!

Frankenfood Frankencar

Strong Public Adoption for Personal Cars But Not GMO Food?

• Risks underestimated or just lower priority

Cars: highly valued personal benefit for independent

transportation and prestige

• Lack of understanding about farming and science

• Public is vulnerable to misinformation tactics of GMO opposition activists

• Risks are overstated

GMO food: benefit of lower food cost

not well appreciated by affluent society

• Fatal Attraction: the intuitive appeal of GMO opposition (Blancke et al., 2015, Trends Pl. Sci. early online version)

• Dr. Camille Ryan, University of Saskatchewan – The Art and Science of the Ag and Food Conversation

Webinar • http://www.agriculturemorethanever.ca/from-the-team/learn-how-

to-speak-up-for-ag/ – Agriculture, Science and Public Perceptions on

Biotechnology, August 2014 report for GenomePrairie • http://www.genomeprairie.ca/files/8214/1265/1492/GP_SCI_COMM_Final_W

eb.pdf

Human cognitive science exploited by GMO opposition

• Often unconscious cognitive predispositions or “intuition” – The core identity of an organism is essential and is

not to be changed • the spiritual belief that man should not modify what

has been created or evolved

• Easy to invoke strong emotions when intuitions are affected – And then dissenting facts are ignored!

• Humans are prone to belief in conspiracies • Seek information that confirms our beliefs and

associate with people with similar biases • We tend to think in pictures

– One picture is worth 1000 peer-reviewed papers

• We are pattern seekers – Scientific method is a relatively new method for

humans to describe and explain patterns – Anecdotes and testimonials have been part of our

psyche as long as magic, myths and stories • Celebrity “experts” and the rise of science “fictation”

What can we do? • Scientist performance is measured in number of peer-

reviewed papers / grants but where is the reward for communicating with public? – It’s not my job… but do you want the NGOs and celebrities to do

it? – Government communication policies

• Personal relationships, social media – Get some training – Start response with your personal values, concerns, priorities

• My example for GMO food purchase – Don’t lecture – facts are secondary, best to provide your

perspective and suggest some alternative sources of information

– Start a blog, vlog • Publish open access papers, transparency of regulatory

submissions