TRANSGENIC PLANTS WITH INCREASED PHYSIOLOGICAL

EFFICIENCY

R.s

Introduction To improve crop yield in a sustainable manner to

meet the demands of increasing world population is a central challenge in today's world.

Some fundamental component of plant productivity which have not been used to select for increased yield is Photosynthesis, Phosphorus uptake efficiency (PUE) , nitrogen uptake efficiency (NUE), water uptake efficiency(WUE).

Through application of Transgene technologies together with novel modeling approaches could help in attaining goal for the welfare of humankind.

Physiological efficiency

In plants physiological enhancements is done by interaction of many factors.

Factors affecting Physiological efficiency: Photosynthesis Nitrogen uptake Phosphorus uptake Water uptake

C3 cycle or Calvin Benson cycle

Majority of plant species fix atmospheric CO2 through C3 cycle using RUBISCO enzyme.

The first stable product of this cycle is PGA (Phosphoglycerate), that’s why called as C3 cycle.

This PGA further forms triose phosphates using ATP & NADPH.

Triose & hexose phosphates are used for the synthesis of amino acids, sucrose, starch etc.

Manipulation of this pathway has the potential to increase yield & production of carbon.

Relationship between the C3 cycle & photorespiratory cycle.

Rubisco-Target enzyme for manipulation of C3 cycle

Major problem of C3 cycle is enzyme RUBISCO which has low turnover number & catalyses: carboxylation & oxygenation rxns.

The oxygenation reaction directs the flow of carbon through the photorespiratory pathway, resulting in loss of 25-30% carbon.

Under high temperature & drought condition there is more increase in oxygenase reaction.

Over the last 20 years, considerable effort has gone into trying to increase the efficiency of Rubisco carboxylase reaction & to reduce losses in yield by eliminating photorespiration.

Transgenic approach to manipulate the environmental responses of the C3 carbon fixation cycle Combination of genetic engg. & transgene technology

have been used to reduce photorespiration. Three approaches:- Two approaches are focused on Increasing the

carboxylation efficiency of Rubisco.i. By reducing the oxygenase reaction directly.ii. By manipulating the rubisco enzyme by concentrating

CO2 in the region of Rubisco through the introduction of enzymes of the C4 pathway.

iii. Reduce photorespiration directly by manipulation of enzymes in this pathway.

a. Introduction of improved Rubisco into transgenic plants

Rubisco protein engineering Rubisco enzyme is composed of eight large

chloroplast encoded & eight small nuclear encoded protein subunits.

I. LSU- contains information for catalysis.II. SSU- function is unknown. Genetic screening & site directed mutagenesis have

been focused on LSU to alter the discrimination between CO2 & O2 i.e. specificity factor.

Random mutagenesis such as DNA shuffling & accelerated evolution have been used to alter the catalytic properties of Rubisco.

But till date these approaches have not resulted in isolation of improved Rubisco enzymes.

• Recently, it is possible to form an active Rubisco invitro using unfolded large subunits with the chaperon proteins GroEL/ES, RBCX & ATP( Liu et al., 2010).

• Recent demonstration of successful expression of Rubisco large & small subunits as a single fusion proteins in the chloroplast ,which assembled into an active holoenzyme provides the opportunity to use expression of foreign Rubisco as a strategy to improve the C3 cycle.(Whitney et al.,2009)

In a recent survey of 24 Rubisco enzymes from mediterranean region has revealed that the Rubisco specificity factor was greater for plants growing in hot, arid or saline envt.

It represents a real opportunity to introduce these into transgenic plants using plastome transformation.

This would avoid the problems of incompatible genetic & protein assembly mechanisms encountered with red algal Rubisco’s & has the potential to improve photosynthesis ,particularly in plants grown in hot, arid conditions.

b. Screening for natural variation in Rubisco catalytic properties

In this approach we screen different species to find improved Rubisco enzyme having high specificity factor.

This enzymes is further introduced into plants through transformation techniques.

In Red alga Gladieria partita & cyanidium caldarium specificity factor is very high.

Rubisco from G. partita when introduced into tobacco plants using plastome transformation.

Significant level of LSU protein were detected in the plant.

Reason could be the requirement for plastid chaperonin proteins, as little is currently known abt the Rubisco assembly process.

c. Genetic manipulation of the photorespiratory C2 cycle.

When reduction of enzymes involved in C2 cycle was brought through antisense approaches there was decrease in other plant activity.

Reduction in glycine decarboxylase activity resulted in lower photosynthesis.

Reduction in serine hydroxy methyl transferase (SHMT), plants were unable to grow in ambient air.

Enzyme D- glycerate 3-kinase catalyses the final step in c2 cycle, converting glycerate to 3-PGA.

An arabidopsis T-DNA insertional mutant in this gene lacks glycerate kinase activity is unviable in air.

C3 photosynthesis is unlikely to be improved by direct manipulation of levels of enzymes.

d. Introducing CO2 concentrating mechanisms into C3 plants

1. Engineering C3 plants to express C4 enzymes

C4 pathway:

a. Evolved in response to increasing O2 levels as a mechanism to increase the Co2 conc. at the site of Rubisco.

b. Plants sp. Utilizing this pathway have advantages like increase in photosynthetic performance, high N2 & water use efficiency.

c. Conventional breeding approaches were proved to be unsuccessful.

Transgenic approaches were used where introduction of C4 enzymes into C3 plants resulted good results.

In Transgenic potato plants when PEPC & NADP-ME were introduced, changes were seen in the compensation point for photosynthesis which implies that photorespiration has been increased.

In another examples, the decarboxylating enzymes PEP Carboxylase (PEPCK) from C4 grass, Urochloa Pancides has been expressed in rice chloroplast & increased amount of carbon were diverted to synthesize C4 acids such as Malate & OAA.

It is clear from this work that it is possible to introduce & express C4 enzymes in C3 plants & this can bring abt. Changes in carbon fluxes.

2. Expression of the cyanobacterial ict B in higher plants

Co2 concentrating mechanisms have been found in algal sp & cyanobacteria.

These adaptation present the possibility to exploit existing solution that have evolved in these organisms to improve C3 plants.

A high CO2 requiring cyanobacterium expresses ict B gene, which is involved in inorganic carbon accumulation.

• ict B gene from Synchococcus PCC7942 when introduced in tobacco plants, there was higher photosynthetic rates under saturating CO2 levels.

• Similar results were obtained in Arabidopsis plants expressing ictB gene from Anabaena sp.

b. Prokaryotic carboxysome provides a microcompartment in the cell transports & fixes Co2 acting as CCM.

c. Eukaryotic algae possess a structure, the pyrenoid which may act to concentrate CO2 at Rubisco.

Both of these CCM’s provide components that could be transferred to higher plants to reduce photorespiration & increase water uptake & nitrogen use efficiency.

However this approach would be complex feat of gene engineering & likely to be successful only in the long term.

Plant,cell & environment,2006

3. Manipulation of Rubisco activation

a. Temperature above 30-35 degree Celsius there is reduction in carbon assimilation because of reversible decrease in Rubisco activation.

b. The activation state of Rubisco is dependent on the enzyme Rubisco activase which is unstable at high temp.

c. Through DNA shuffling which is random mutagenesis strategy, a thermostable rubisco activase was produced.

d. Which was further introduced into an arabidopsis Rubisco activase null mutant line.

e. A two fold increase in leaf area & a 30% higher photosynthetic rates were obtained compared to wild plants.

f. This finding gave clear evidence of potential of this approach to maintain photosynthetic capacity during high temp.

e. Another approach- Limit the factors involved in C3 photosynthesis

Transgenic studies has shown that Rubisco doesn’t limit the C3 cycle in all conditions & that enzymes of regenerative phase of the cycle also play a role in determining the rate of photosynthesis.

Increasing regenerative capacity of the C3 cycle Regeneration of CO2 acceptor, RUBP requires 8 enzymes. Overexpression of SBPase (sedoheptulose- 1,7-

bisphosphate),FBPase, transketolase & aldolase in transgenic plants resulted in increased photosynthetic CO2 fixation.

It would be interesting to engineer plants with increased amt of one or more enzymes in the regenerative phase of the C3 cycle as these may allow plants to take better advantages of high CO2 conditions, particularly in low nitrogen conditions.

f. Design & introduction of synthetic carbon fixation pathway In nature there are 5 other Co2 fixation

pathways found in Archaea (Berg et al., 2010).

These organisms don’t use either Rubisco or PEP carboxylase as carboxylation enzyme.

This raises the possibility of constructing a novel CO2 fixation pathways that may be more efficient than C3 cycle.

This approach may offer the solution to the problem of Rubisco, without having to re-design plant .

Nitrogen uptake efficiency (NUE)

NUE includes N uptake, utilization, or acquisition efficiency, expressed as a ratio of nitrate uptake efficiency & utilization efficiency.

Plants physiological efficiency depends upon NUE by PE = (Gwf –Gwc)/Nf uptake–Nc uptake There is significant interest in genetic engineering

crops to improve NUE. N assimilatory pathway is one of the most widely

chosen targets for improvement of NUE. Targets of NUE are: Nitrate transporters, nitrate reductase (NR), nitrite

reductase (NiR), plastidic glutamine synthetase (GS2), and ferredoxin glutamate synthase (Fd-GOGAT), cytosolic glutamine synthetase (GS1), reduced nicotinamide adenine dinucleotide glutamate synthase (NADH-GOGAT), glutamate dehydrogenase (GDH),

GENETIC ENGINEERING APPROACHES TO IMPROVING NITROGEN USE EFFICIENCY 1. Engineering plants with transport gene systems • Crop plants obtain N from the soil primarily as nitrate or ammonia & this uptake occurs by the specific transporters located in root cell membrane.• Nitrate is reduced to ammonium through the combined action of nitrate reductase (NR) and nitrite reductase (NiR). • The overexpression of either the NR or the NiR gene in plants increases mRNA levels and often affects N uptake.

2. Manipulating Genes of N Uptake • N uptake is the first step in nitrate assimilation,Two types of nitrate uptake systems:• Low-affinity transport system encoded by NRT1 gene family and high-affinity transport system encoded by NRT2 gene family.• Overexpression of high-affinity nitrate/nitrite transporters in tobacco and Arabidopsis reported enhanced nitrate uptake.

3. Manipulating Genes of N Assimilation

• Primary nitrate assimilation involves NR, NiR, plastidic GS (GS2), and Fd-GOGAT, while cytosolic GS (GS1) and NADH-GOGAT are part of secondary ammonia assimilation and remobilization.

• Overexpression of GS2 has also been reported in rice and tobacco, but no significant improvement in terms of NUE was observed.

• Overexpression of GS1 gene has been tried in several plants such as wheat, tobacco, and maize have resulted in higher grain yield and biomass with improved N content.

• Transgenic overexpression and anti-sense technology has been employed to modulate the expression of NADH-GOGAT gene in rice and alfalfa plants.

• Overexpression of  OsNADH-GOGAT1 in transgenic rice under the control of its own promoter showed an increase in spikelet weight.

4. Manipulating Genes of N Translocation and Remobilization

AS (aspartate synthase encoded by Aln1 gene) is involved in N remobilization & proposed as one of the key steps in improving NUE in plants.

Genetically engineered rice (Oryza sativa L.) was developed by introducing barley alanine aminotranferase complementary DNA (cDNA) driven by tissue-specific OsAnt1 promoter.

These plants showed improved nitrate content confirming increased NUE.

Overexpression of alanine aminotransferase (AlaAT) gene in rice under the control of a tissue-specific promoter showed a strong NUE phenotype.

Though the importance of Glutamate dehydrogenase (GDH) in higher plant N remobilization is still controversial, transgenic plants overexpresing gdhA gene were shown to have improved amino acid content, higher yields in maize and wheat.

More recently, Arabidopsis NRT1transporter NRT1.7 has been implicated in nitrate remobilization from source to sink tissues by mutant analysis.

5. Manipulating Genes of C Metabolism Transgenic Arabidopsis plants overexpressing STP13, a

member of sugar transporter family, showed increased rates of glucose uptake, higher internal sugar levels, and more total C per plant.

STP13OX seedlings also displayed improved N use, with the induction of a nitrate transporter and higher total N per plant.

6. Manipulating Signaling Targets Nitrate is a potent signal that affects N and carbon metabolism. Identification of nitrate-responsive gene expression profiles of

plants would offer potential targets for improvement of NUE. Recently, bioinformatic approaches have been employed to

search for TFs in nitrate-responsive genes. TFs such as AtNF-YB1 from Arabidopsis thaliana and ZmNF-

YB2 from maize improved NUE and water use efficiency (WUE) in transgenic plants.

Phosphorus uptake efficiency (PUE)

In plants, P plays a pivotal role in photosynthetic regulation, energy conservation, and in carbon metabolism.

Plants possess a large number of adaptive responses to Pi (orthophosphate) limitation that provide potential raw materials to enhance Pi scavenging abilities of crop plants.

Understanding and engineering these adaptive responses to increase the efficiency of crop capture of natural and fertilizer Pi in soils is one way to optimize Pi use efficiency (PUE).

Research on the molecular and physiological basis of Pi uptake is facilitating the generation of plants with enhanced Pi use efficiency by genetic engineering.

• Over-expression of the Pi transporter gene Pht1:1 in tobacco cell cultures resulted in an increased rate of Pi uptake. • Unfortunately this enhanced uptake capacity has not been confirmed at the whole plant level.

• A rice transcription factor (TF) (OsPTF1) involved in the phosphate starvation response has been reported (Yi et al., 2005). • OsPTF1 is expressed in phloem cells of the primary root, leaves and lateral roots. • Overexpression of OsPTF1 enhances rice tolerance to Pi starvation. • Interestingly, microarray data on this OsPTF1 transgenic rice plants showed a concomitantly enhanced expression of rice H+-PPase.

Improving PUE by up-regulation of a type I H+-Pyrophosphatase (plant proton-translocating pyrophosphatases) • Plants have two phylogenetically distinct H+-PPases: type I and type II. Type I H+-PPases depend on cytosolic K+ for their activity and are moderately sensitive to inhibition by Ca2+ and type II H+-PPases are K+-insensitive but extremely Ca2+-sensitive.• Overexpression of the type I H+-PPase AVP1 in Arabidopsis (AtAVP1OX) resulted in enhanced salt tolerance and drought resistance (Gaxiola et al., 2001).

• Overexpression of an H+-PPase gene from Thellungiella halophila in cotton enhances salt tolerance and improves growth and photosynthetic performance.

• Another interesting approach was to engineer A. thaliana plants with an alfalfa (Medicago truncatula) phytase gene. These transgenic plants displayed an improved acquisition capacity of organic phosphorus when grown on phytate as the only source of phosphorus (Xiao et al., 2005).

• Similarly, Nicotiana tobaccum plants engineered to express a fungal phytase gene resulted in improved phosphorus nutrition when grown in amended soils (George et al., 2005).

Water use efficiency (WUE)

WUE is briefly defined as the leaf WUE (leaf photosynthetic rate per transpiration rate), whole plant WUE (the ratio of above ground biomass or dry matter per unit area, and water use by crop); yield WUE (crop grain per unit area, to the transpiration loss from the crop).

The transgenic relating to high WUE

Drought resistance and high WUE has close relationship in crop breeding.

Some drought-resistant genes are used to improve WUE in crop.

Transgenic lines expressing the HVA1 gene having higher WUE values was indicated .

Transgenic wheat plants constitutively expressing the barley HVA1 gene in response to soil water deficits have increased growth conditions.

The ERECTA gene has been isolated from Arabidopsis and found it can regulate transpiration efficiency and affect stomatal density,epidermal cell expansion, mesophyll cell proliferation and cell–cell communication.

• Increasing ABA production under optimal conditions by overexpressing SlNCED1 has been shown to reduce stomatal conductance and improve WUE in Tomato plants.

• In Tobacco Aquaporin1 is protein which increases the permeability of membrane to water. •Overexpression of NtAQP1 in tomato (Solanum lycopersicum) plants (TOM-NtAQP1) resulted in Improved water use efficiency, higher stomatal conductance, whole-plant transpiration.

These results indicated that transgenic method is a promising way for improving crop WUE.

•In recent years, many new methods appeared providing powerful tool for genetic improvement of WUE in crops, such as T-DNA insertions and deletion mutants are currently used as a tool to identify phenotypic mutants for characterizing relevant genes at the molecular level and reveal their functions.

•Suppression subtractive hybridization and microarray-based expression profiling are used to isolate and identify candidate genes.

•Gene-network estimation method to facilitate analysis of gene interactions can serve as specialized tools in bioinformatics, which open new possibilities for the development of new cultivars with higher WUE

Conclusion Manipulation of C3 cycle offers an

opportunity to increase photosynthesis yield.

Improvement of C3 cycle is not just about increasing CO2 fixation but should also aim to increase both nitrogen use efficiency & phosphorus use efficiency.

Despite the plethora of data available on Rubisco ,it seems unlikely to engineer a improved Rubisco.

Through transgenic technologies it has been possible to manipulate the physiological efficiency of plants.