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ENGINEERING OF C3 TO C4 RICE

The relationship between rice The relationship between rice production and population for production and population for Asian rice consumers (1961-2004)Asian rice consumers (1961-2004) Data source: UN and FAOData source: UN and FAO

The production of

C4 RiceC4 rice could:• Increase rice yield by 50%• Double water-use efficiency• Improve nitrogen use efficiency• Increase 50% radiation use efficiency

C4 photosynthesis is one of the few evolutionary mechanisms that could deliver these superior combination of beneficial traits.

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C4 rice would confer benefits on all of the global rice eco-systems

Rice Almanac (Maclean et. al. 2002)

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C4 supercharges photosynthesis using a two compartment CO2 concentrating mechanism

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C4 Plants: C4 plants have the greater rate of CO2

assimilation than C3 plants. - PEP carboxylase has great affinity for CO2.

- C4 plants show little or no photorespiration compared to C3 plants. C4 plants are more adaptable to

environmental stress than C3 plants. CO2 fixation in C4 photosynthesis requires

more ATP than C3 photosynthesis.7

Evolution of C3 to C4 Plants• When plants first evolved, photorespiration

was not a problem due to the low level of O2 and high CO2 concentration in the environment.

• As a by-product of photosynthesis, O2 accumulated in the atmosphere and reached the present level million of years ago.

• Current atmospheric CO2 conc. limits C3 photosynthesis. Furthermore, photorespiration reduces net carbon gain and productivity of C3 plants as much as 25%.

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•With modification in leaf anatomy, some tropical species (e.g.- maize, sugarcane) have evolved a biochemical “CO2 pump” the C4 photosynthetic pathway, to concentrate atmospheric CO2 and thus overcoming photorespiration.•C4 plants thus exhibit many desirable agronomic traits: high photosynthesis rate, fast growth, high water & mineral use efficiency.

The Timeline for C4 Rice

It will likely take a minimum of 15 years of coordinated research carried out in the laboratories of C4 Consortium to deliver C4 rice to plant breeders in the developing world. ( The C4 Rice Consortium, IRRI)

Building The C4 Machinery

A.) Characterize Regulatory controls:-

Identification of-• Regulatory Elements (REs): i.e. ‘cis’-regulatory elements & ‘trans’-regulatory elements.• Transcription Factors (TFs): include GLK1, GLK2 genes in MYB family, HY5 family, DOF family proteins.• microRNAs• Promoter Elements (PEs): allow cell-specific expression & leaf-developmental gradient profiles in rice.

Source: C4 Rice Consortium, IRRI

Identify promoter elements to allow cell specific expression in rice (C4 Rice Consortium, IRRI)

B.) Anatomical change:- C3 to C4 leaf anatomy change in 2 ways- 1.) Krantz anatomy development: - increase the no. and size of chloroplasts in BSC (bundle sheath cells) in rice. (e.g.- over expression of GLK genes using Z. mays PEPC promoter for MC (mesophyll cell) & Z. japonica PEP-CK promoter for BSC specific expression.) - reduce vein spacing thereby increasing the vein density in the leaf in rice.

Source: C4 Rice Consortium, IRRI

A) C3 ( O. sativa L., IR64 var.) leafB) C4 ( Setaria viridis) leaf-MC of rice has more chloroplasts than its

BSC.-In C4 leaf, chloroplasts has localized in BSC

as well as in MC.

2.) Metabolome alteration: - C4 photosynthetic metabolism and anatomical features of leaf influence each other. e.g.- manipulating enzymes in the Calvin cycle, including RuBisCO, SBPase and CP12 altered leaf anatomy.

C.) Biochemical change:- Alteration of biochemical activities are based on-

a.) Single-cell model: Burnell, 1990 Mesophyll cells only:- -Induce expression of CA (carbonic anhydrase), PEPC (PEP carboxylase) in MC cytosol; NADP-MDH (NADP malate dehydrogenase), NADP-ME/PEP-CK (PEP carboxykinase), PPDK (pyruvate ortho-phosphate dikinase) in MC chloroplast. -Reduce expression (in chloroplast) of CA, GDC (Glycine decarboxylase, to reduce photorespiration).

Single celled C4 photosynthesis (Burnell, 1990)Enzymes: 1.- spinach CA; 2.- maize leaf PEPC; 3.- Urochloa PEP-CK; 4.- maize leaf PPDK.

b.) Dual-cell model: • Gene expression in- Mesophyll cells:- a.Reduction of Calvin cycle activity, photorespiration;b. Reduce expression of RuBisCO, GDC;c. Induce expression of CA, PEPC in cytosol & PPDK, NADP-MDH in chloroplast. Bundle sheath cells:- a.Induction of Calvin cycle activity;b.Induced expression of RuBisCO, GDC, PEP-CK, NADP-ME.

NADP-ME type C4 pathway-Decarboxylation occurs in BSC chloroplast.

NAD-ME type C4 pathway-Decarboxylation occurs in BSC mitochondria.

PEP-CK type C4 pathway -Decarboxylations occur both in BSC cytosol & mitochondria.

• Transporters expression-

- Specific transporters identified through proteomics in maize BSC & MC are transformed into indica rice variety.

- Transporters are OMT1 (2-oxoglutarate/malate transporter), DiT2 (dicarboxylate transporter2), PPT1 (PEP/phosphate transporter), MEP (mesophyll envelop protein), TPT (triose-phosphate phosphate translocator).

Transporters study of NADP-ME subtype C4 photosynthetic pathway i.e. being genetically engineered in indica rice variety by the C4 Rice Consortium, IRRI.

c.) RuBisCO activity alteration:

-Better RuBisCO: higher specificity for CO2. -Protect RuBisCO from O2: confine to chloroplast without PSII.

A theoretical framework of the potential mechanisms underlining development of C4 photosynthesis.

Work in Progress by the C4 Rice Consortium, IRRI

Introduction of C4 genes in indica rice from various crops:• Maize- pepc gene ppdk gene nadp-me gene rbcS gene• Echinochloa- ppdk gene• Sorghum- nadp-mdh gene• Urochloa panicoides- pepck gene

Methods employed for gene transfer into rice:

• Electroporation

• Particle Gun Method of Gene Transfer Or, Biolistic Method of Gene Transfer

• Experimental Procedures•Plasmid construction- -pepc- and rbcS-GUS fusion genes were cloned into

pUC19 vector. -35S promoter of CaMV and GUS gene into pBI221.•Transformation, in-vitro culture & selection of transgenic•PCR and Southern Blotting analysis•Hybridization analysis, DNA gel-retardation assay, DNA

microarray•Total RNA isolation, Northern Blotting analysis•RT-PCR and systematic reverse genetic screening•Protein extraction & Western Blot analysis•SEM analysis•Measurement of starch, lipid & total protein contents

Source: C4 Rice Consortium, IRRI

Some interesting rice mutants are starting to emerge

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Transgenic plants showing normal phenotype and good seed setting like control plants.

Sorghum mutant with increased vein density & mesophyll cells between veins

Limitations

• The commercialization of transgenic rice is still difficult in the present era.• C4 rice research is very expensive & laborious owing to

huge distance of antimony and genetics between C3 and C4 rice.• Although over expression of C4 genes in rice showed

diverse effects, it is still far from the purpose of increasing yield greatly.• Although some changes in the photosynthetic apparatus

have been recorded in transgenics but no significant direct increase of photosynthesis rate was recorded apart from alterations in stomatal conductance.

Future Prospects

• Recent studies suggest that CO2 level is rising at slightly less than 2 ppm per year. In the next 50 years it is estimated in rising by an average of 3 ppm per year. By 2050, the atmospheric CO2 level will be 520 ppm that may not be high enough to allow C3 plants yielding more than C4 plants, even with the C3 plants that are adapted in the high CO2 levels.

• Hence engineering of C4 rice would be beneficial to meet the production goal within the next half-century.

Conclusion• Engineering the C4 photosynthetic pathway into C3 plants

has the potential to dramatically increase the yields & correspondingly plays a crucial role in ensuring future global food security.• C4 rice project will not only enhance our understanding

of molecular mechanisms underlining C4 development, the basic design of an ideal C4 system but also develop many biotechnological tools, all of which will help engineering C4 wheat, C4 cotton, or even C4 forestry.• As a highly interdisciplinary project, the C4 rice project

requires collaborations among scientists from diverse disciplines & more work is needed in order to convert less efficient C3 rice to more efficient C4 rice.