Course Seminaron

Conversion Of C3 To C4 Plants

Supervisor: Prof. R. K. Singh

Presented by:Dilruba A BanoID No: G-12092

Department of Genetics and Plant BreedingInstitute of Agricultural Sciences

Banaras Hindu University, Varanasi

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Photosynthesis

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Introduction

C3 plantsPlants that produces the 3 carbon

compound phosphoglyceric acid at

the first stage of photosynthesis

C4 plants Plants in which the CO2 is first fixed into a compound containing 4 carbon atom before entering the Calvin cycle of photosynthesis

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C3 Plants

Most of our conventional crops, assimilate

atmospheric CO2 by the C3 pathway of

photosynthesis. Photosynthetically, these plants are

underachievers because, on the one hand, they

assimilate atmospheric CO2 into sugars but, on the

other hand, part of the potential for sugar

production is lost by respiration in daylight,

releasing CO2 into the atmosphere, a wasteful

process termed as photorespiration.

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This is due to the dual function of the key

photosynthetic enzyme , ribulose 1,5-

bisphosphate carboxylase / oxygenase

(Rubisco).

CO2 favours the carboxylase reaction and thus

net photosynthesis; whereas high O2 promotes

the oxygenase reaction leading to

photorespiration.

CONTD...

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Photorespiration When Rubisco reacts with O2 instead of CO2

Occurs under the following conditions:– High O2 concentrations– High heat

Photorespiration is estimated to reduce photosynthetic efficiency by 25%

At high temperature, plants close their stomata to conserve water

They continue to do photosynthesis use up CO2 and produce O2 creates high O2 concentrations inside the plant photorespiration occurs

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C3 to C4 Plants

When plants first evolved, photorespiration was not a problem because the atmosphere then was high in CO2 and low in O2.

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

Current atmospheric CO2 levels limit photosynthesis in C3 plants. Furthermore, photorespiration reduces net carbon gain and productivity of C3 plants by as much as 40%.

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With modifications in leaf anatomy, some tropical

species (e.g., maize and sugarcane) have evolved

a biochemical “CO2 pump”, the C4 pathway of

photosynthesis, to concentrate atmospheric CO2

in the leaf and thus overcome photorespiration.

C4 plants thus exhibit many desirable agronomic

traits: high rate of photosynthesis, fast growth

and high efficiency in water and mineral use.

Contd….

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C4 Plants 1. C4 species have greater rate of CO2 assimilation

than C3 species

a. PEP carboxylase has great affinity for CO2

b. C4 plant show little photorespiration as compared

to C3 plant resulting production of dry matter

2. C4 plants are more adapted to environmental

stress than C3 plants.

3. CO2 fixation require more ATP than C3 plants.

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How does the C4 Pathway limit photorespiration?

Bundle sheath cells are far from the surface–

less O2 access

PEP Carboxylase doesn’t have an affinity for

O2 allows plant to collect a lot of CO2 and

concentrate it in the bundle sheath cells.

(where Rubisco is)

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Differences between C3 & C4 cycle

Character C3 plant C4 plant

CO2 acceptor First CO2 acceptor is

Ribulose 1,5

diphosphate

First CO2 acceptor is

phosphoenol

pyruvate (PEP)

First stable

product

Phosphoglyceric acid Oxaloacetate (OAA)

Type of

chloroplast

One type Dimorphic chloroplast

are present in which

kranz anatomy

found

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Optimum

temperature

for process

10-250C 30-450C

CO2 compensation

point

50-150ppm 0-10ppm.

Photorespiration Present and easily

detectable

Absent or present

to a slight degree

Contd.

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Examples

C3 – Rice, wheat, potato, sugar beet.

C4 – sugarcane, maize and sorghum.

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C4 discovered by M.D. Hatch & C.R. Slack

C3 cycle (Calvin & Benson cycle)

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Net Photosynthetic rate V/S light Intensity

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Quantum yield of photosynthesis with increase in temperature

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Engineering for C4 traits

There are no closely related C3 and C4 crops that

we can use to transfer the C4 traits to C3 crops by

a traditional breeding approach. Thus genetic engineering is the best alternative.

In engineering C4 photosynthesis, there are two

important components to be considered:

1. Specialized leaf structure

2. Biochemical pathway (enzymes)

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Our first goal is to engineer the key enzymes

involved in C4 photosynthesis without

manipulating Kranz leaf anatomy.

Identification of single cell C4 system

in Hydrilla sp. and terrestrial chenopods such

as Borszczowia aralocaspica, where all the

C4 photosynthetic features are present without

Kranz anatomy and it is considered that

development of C4 cycle is possible in the

single cell type.

Cont….

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Why this is needed?

Reduce Global warming

Increase photosynthesis rate

Increase light use efficiency

Increase water use efficiency

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C4 RicePEPC and PPDK transgenic rice plants exhibit higher photosynthetic capacity than untransformed plants, mainly due to an increased stomatal conductance (i.e., more atmospheric CO2 becomes available for fixation).

Preliminary field trials show 10-30% and 30-35% increases in grain yield for PEPC and PPDK transgenic rice plants, respectively.

The activities of PEP carboxylase in leaves of some of these transgenic rice plants were two- to threefold higher even than those in maize.

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Increased synthesis of organic solutes (e.g., malate) by the enzymes in the guard cells may be responsible for the enhanced conductance of CO2

by the stomates.

C4 cycle-related genes have been introduced into rice including PEPC, pyruvate orthophosphate dikinase (PPDK) gene, phosphoenolpyruvate carboxykinase (PEPCK) gene, NADPmalic enzyme gene (ME) gene and NADPmalate dehydrogenase (MDH) gene.

Cont…

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Introduction of C4 genes in rice from various crops

Maize

PEPC

PPDK

NADP – Malic enzyme (ME) Echinochloa - PPDK Sorghum – MDH Urochloa panicoides - PCK

Methods Employed for gene transfer

• Agro-bacterium mediated transformation

• Biolistic transformation

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Procedure for engineering indica rice expressing pepc gene from maize using biolistic transformation

1. Plasmid construct, plant material, transformation and in vitro culture

2. Polymerase chain reaction (PCR) and Southern blot analysis

3. Total RNA isolation and Northern blot analysis

4. Western blotting

5. Measurement of photosynthesis and agronomic data

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Schematic diagram of the intact maize pepc gene.

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

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Achievement in Rice crop Converting the photosynthesis of rice from the less-

efficient C3 to C4 form would increase yields by 50%.

In countries where billions of poor people depend on

rice as their staple food, benefits of such an

improvement would be immense in the face of

increasing world population, increasing food prices

and decreasing natural resources.

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Limitations

The commercialization of transgenic rice is still difficult in the present times.

Although over expressing these C4 -related genes in rice showed diverse effects, it is still far from the purpose of increasing the yield greatly, and even lead to severe negative effects.

C4 rice research is very laborious due to huge distances of antimony and genetics between C3 and C4 plants.

Although some changes in photosynthetic characteristics have been recorded in transgenics with respect to the nontransformants but no significant direct increase of photosynthesis rate was recorded apart from some alteration in stomatal conductance.

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Future Prospects

Currently, CO2 levels are rising at slightly less than 2 ppm per year. If we take for granted they will raise over the next 50 years by an average of 3 ppm per year, then the atmospheric CO2 level will be 520 ppm by 2050. This may not be high enough to allow for C3 plants to yield more than C4 plants, even with C3 plants that are adapted to higher CO2

Hence engineering a C4 plant would be beneficial to meet production goals within the next half-century.

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Conclusion C4 plants are more productive than C3 plants when they are grown

under their respective optimum conditions.

C4 plants exhibit higher water and nitrogen use efficiencies compared to C3 plants, which results in an increased dry matter production.

Concentrating CO2 at the site of Rubisco allow engineered C3 plants to reduce stomatal conductance under drought conditions without a dramatic decline in the rate of CO2 assimilation.

This allows both the use of new areas for crop production required for feeding the growing world population and the reduction of inputs into the system (such as fertilizers) and also conserving natural resources. These factors are much more relevant to today’s necessities than the mere increase in biomass production.

More work is needed in order to convert the less efficient C3 to more efficient C4 plants.

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