The Twin Tale

The latter part of the 1960s marked two very important events that continue to have a recurrent bearing on the global food security. One of them was characterized by the use of short-statured so-called high yielding varieties and it went on to increase the food production by 10 folds. While all this, popularly summarized as green revolution, was going on at the same time a vibrant new research field was ignited by the discovery that instead of fixing CO2 into a 3 carbon compound some plants proximally fix CO2 into a four-carbon compound ( Hatch and Slack 1966, Hatch 2002) and this concludingly marked the birth of C4 photosynthesis. In the decades that followed, scientists discovered the underlying mechanisms how the pathway was partitioned between two morphologically different cell types and thus much biochemistry of C4 photosynthesis was unravelled.  However, virtually nothing was known about the genetic patterns that underpinned this biochemistry. This was a time when the green revolution cultivars reached a yield barrier and that too at a time when 548 million people worldwide were undernourished. This was also the time when most of the C4 models were displaying recalcitrance towards genetic manipulation and the scientists thought was limited by non-availability of high throughput ‘omic’ sequences. However, over the latter half of the last decade, there has been resurgence regarding this and hopes are green again for a second green revolution. By 2050 there would be 1309 Million Tonnes demand of rice and C3 rice even when best managed can yield only 915Million tonnes. (C4C data) This gap of 394MT between the supply and demand of food grains and this could only be resolved if our rice cultivars are performing the naturally selected better variant of photosynthesis i.e. through the C4 pathway.

Keywords: C4 Rice, Food Security, Transcriptome Analysis

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Figure-1  Caemmerer, V. (2017), The C4 Rice Project, Retrieved from

Tit-Bits of Supercharged Photosynthesis ‘The C4 Pathway’

In the normal C3 cycle (elucidated by Calvin and Benson in 1956) n cycled to yield a molecule of sugar and at the same time regenerating the RUBP initially used. This is accomplished by a battery of enzymes which utilize the ATP and reducing equivalents generated earlier using sunlight.the Carbon dioxide that has been taken by plants combines with RUBP or Ribulose 1,5 Bisphosphate in presence of enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to fix the carbon dioxide into a three-carbon compound 3-phosphoglycerate (3PGA) which is then cycled to yield a molecule of sugar and at the same time regenerating the RUBP initially used. This is accomplished by a battery of enzymes which utilize the ATP and reducing equivalents generated earlier using sunlight.

But all is not so sunny. There is a big issue when the operating conditions have the high light intensity or temperature or both. Under such conditions Rubisco instead of showing carboxylase activity it shows oxygenase activity resulting into a phenomenon called ‘Photorespiration’ where like us plants also start respiring and releasing carbon dioxide instead of oxygen and the total amount of photosynthate produced by plants decreases or technically speaking yield decreases. This loss termed as ‘photorespiratory carbon loss’ is the fundamental reason behind C3 rice more than 85 percent of which is grown in tropical areas where high temperature and high light intensity prevail during most part of the growing season.

However certain plants called the C4 plants have a different mechanism altogether to reduce this photorespiration. Instead of fixing carbon dioxide into 3PGAthey fix it into a four-carbon oxalo acetic acid (OAA) using phosphor-enol-pyruvate (PEP) instead of RUBP and then it is transported to a different cell other than the default mesophyll cell.  This cell is called bundle sheath cell and here most of the photosynthetic machinery is present. The OAA that is brought here is decarboxylated to give a 3 carbon compound pyruvate and a carbon dioxide molecule is released into the cell. The repetition of this process builds a high gradient of Carbon dioxide near the Rubisco present in bundle sheath cell and hence whatever be the temperature or light intensity the oxygenase activity of Rubisco is never activated and photorespiration is minimized to negligible levels. Although this requires a relatively higher amount of energy and a more complex battery of transporters, still it is worth it. Further, there is a special arrangement of the two type of cells the bundle sheath cells and the mesophyll cells in the form of the wreath and this morphologically distinct anatomy have been termed as Kranz anatomy.

Sudershan 3

Figure 2: A brief overview on C4 photosynthesis (n.d), Retrieved from

C3 to C4 the proof of concept

Is it possible to actually manipulate the nature in so intervening manner? One hint that we get from studying the evolutionary history of plants is that the C4 pathway arose multiple times independently from the ancestral C3 pathway (Sage et. al. 2011). A closer glance reveals that all the 4600 species of C4 grasses belong to a single PACMAD clade (evolutionary group) and it is tempting to speculate that a preconditioning event occurred in the last common PACMAD ancestor and it may not be a coincidence that low levels of carbonic anhydrase the first enzyme of the C4 shuttle are a characteristic of the entire clade (Langdale, 2011). The fact that C3 system is default is suggested by single-celled C4 systems and facultative C4 plants. In single-celled C4 systems (e.g. Binertia and Suaeda) the fixation of carbon and its cycling are carried out in two different compartments of a single cell rather than in two different bundle sheath and mesophyll cells. In such systems initially, the C3 cycle is operative until the cell forms two compartments and then carries on with the C4 type photosynthesis.(Ueno et al., 1988) Further in facultative C4 plants like Eloechris vivipara and Flaveria brownie have C3 anatomy in submerged leaves and C4 anatomy in aerial leaves.

Rationale for bioengineering C4 rice

Based upon the evolutionary history gathered following two approaches have been concluded

  1. Incremental Gain Hypothesis- C4 photosynthesis evolved gradually
  2. Master Switch Hypothesis- C4 photosynthesis evolved through a common mutational event

The first hypothesis suggests that small C4 favouring mutations occurred over time and got pyramided over and over into the genome while the second suggests a single large mutation that may have occurred in the common ancestor and let to the overall transition of a C3 plant into the C4 plant.

Another important morphological parameter is vein density. It has been found that C4 plants have a very large vein density. This equates to veins (V) being separated by only four photosynthetic cells( bundle sheath ‘BS’ and mesophyll ‘M’ ) in C4 leaves as opposed to up to 20 cells in C3 leaves. As such the repeating unit V-BS-M-M-BS-V of Kranz anatomy is generated.

image 4Figure: 3  Transverse Leaf Sections and Corresponding Schematics of C3 Rice and C4 Maize. Rice (left) and maize (right). Bars = 30 mm. (Langdale, 2011

Requirements to convert a C3 into a C4?

  • A compartment to concentrate CO2 around Rubisco
  • An active light-driven CO2 fixation system
  • Enough supply of photosynthetic energy
  • A pool of metabolites and transporters
  • Characteristic system to decarboxylate metabolic intermediates
  • Prevention of leakage of intermediates as well as CO2
  • Correct stoichiometry of M to BS cells, typically 1:1 in C4 plants

Biotechnological Approaches for Engineering C4 Rice

Testing C4 Mutants

The approach is to mutate C4 plants (particularly Sorghum and Setaria viridis)  and randomly hit some of the C4 characteristics and then try to identify the responsible genetic factors.  Individual plants are mutated using EMS (ethyl methyl sulfonate) or gamma rays. The number of non-specific mutations is reduced by backcrossing and the single nucleotide polymorphisms are genotyped and cross-referenced with sorghum BTx623 that is publicly available (Paterson 2009).

Rice DNA activation tagging

The most direct method in functional gene discovery is to look for a correlation between phenotype and genotype within a specific mutant. This involves overexpressing certain rice genes and then look for C4 like characteristics. T-DNA or transposable elements complexed with reporter genes like GUS (beta-glucuronidase) or GFP (green fluorescent protein) are used which act as tag also to indicate the location of the mutation in the genome which can then be cross-referenced with databases like OryGenes DB, Gramene.(Rizal et al 2012).

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Figure 4: The Twin Tale

Transcriptome analysis (Microarrays and RNA-seq)

A transcriptome is the sum total of functional mRNA transcripts as well as the regulator network within a cell. Comparative transcriptome analysis can help us identify genes that have been turned on or turned off during the transition from the default C3 pathway to C4 pathway. Using microarrays (DNA chips and RNA-seq transcriptome analysis ofC3 and C4 leaves, Other C3 and C4 tissues, C4 BS and M cells, C4 plants at different stages as well as of C3 and C4 plants at comparable developmental stages have been done and a large number of cis-elements regulating this transition at transcriptional level have been identified(Wang et al 2016) 

Epigenetic approaches

It has been found that chromatin configuration also has a bearing on the transition from C3 to C4 rice and hence the effect of histone modification and role of micro RNAs (miRNAs) has been studied in this regard. It is believed that activation of certain histone lysine residues after their acetylation potentiate this transition.

Current biotechnological achievements

  • There are certain transporter proteins whose production needs to be upregulated if this transition from C3 to C4 has to occur
  • There is differential up-regulation of regulatory proteins like ARF2(auxin response factor2), GLK1(golden 2 like 1)and GLK2(golden 2 like 2), Sigma 70 like factors SIG1 and SIG5 as well as chloroplast positioning proteins like GC1(Giant chloroplast 1) and CHUP1 (chloroplast unusual positioning 1)
  • There has been Identification of candidate sequences like MEM1(mesophyll expression module1), and 5’ & 3’ UTRs (untranslated regions)of genes encoding PPdK(pyruvate orthophosphate dikinase) and CA(carbonic anhydrase) the two important enzymes of the C4 These are called candidate sequences as these can lead to cell-specific expression without the need for any promoter sequence. For example MEM! Gets expressed cell specifically in M cell and PPdk and CA in BS cell
  • It has been found that about half of the genes contributing to ATP production are up-regulated
  • ABA and auxin stage-specific relocation has a putative role – in facultative species
  • Scientists have rejected the ‘Master Switch’ hypothesis- based upon expression difference between C4 cycle genes and putative C3 orthologs
  • It has been confirmed that misbalance in nitrogen metabolism between BS and M cells is a pre-requisite for evolution o0f C4 pathway

How close are we?

Evidence of stability of introduced C4 genes into C3 plants particularly rice and tobacco have been reported more frequently now. In rice, several C4 genes have been successfully transformed from maize (which is a C4 plant) and other closely related C4 species that have stably integrated into its genome and expressed over several generations. We have T3 (third generation transgenic) rice plants harbouring PPdK and PEPC (phosphor enol pyruvate carboxylase) genes from maize that have been constantly expressing ZmPPdK (Zea mays PPdk) and ZmPEPC proteins over three consecutive generations (Rizal et. al 2012). The current level of production can sustain the population for 2 and a half decades at the maximum after which our population will outnumber our production. However, given the access to advanced technologies, sustainable funding and the intensive media coverage two decades will be enough time to produce C4 rice. The first phase that was of infrastructure and molecular tool development has been completed and the target in coming years is to transform C3 rice with novel C4 genes and pyramid all C4 genes into a prototype. Biotechnologists are ready with their backpacks to face the immediate need of next green revolution.


  1. Langdale J A (2011). C4 cycles: past present and future research on C4 photosynthesis. The Plant Cell 23:3879-3892
  2. Wang P, Vlad D and Langdale J A (2016). Finding the genes to build C4 rice. Current opinion in Plant Biology. 31:44-50.
  3. Rizal G, Karki S, Thakur V, Chatterjee J, Coe R A, Wanchana S and Quick W P (2012) Towards C4 rice. Asian Journal of Cell Biology. 13: 12-31
  4. Hatch, M.D. (2002). C(4) photosynthesis: Discovery and resolution. Res. 73: 251 256.
  5. Hatch, M.D., and Slack, C.R. (1966). Photosynthesis by sugarcane A new carboxylation reaction and the pathway of sugar formation. Biochem. J. 101: 103–111.
  6. Bassham, J.A., Barker, S.A., Calvin, M., and Quarck, U.C. (1956). Intermediates in the photosynthetic cycle. Biophys. Acta 21: 376–377.
  7. Sage, R.F., Christin, P.-A., and Edwards, E.J. (2011). The C(4) plant lineages of planet Earth. J. Exp. Bot. 62: 3155–3169.
  8. Ueno, O., Samejima, M., Muto, S., and Miyachi, S. (1988). Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: Expression of C(4) and C(3) modes in contrasting environments. Natl. Acad. Sci. USA 85: 6733–6737
  9. Paterson et al. (2009). Sorghum bicolor genome and diversification or grasses. Nature, 457:551-556

Authored By:

Sudershan Mishra

M.Sc. (Ag) Previous

Department of Plant Physiology,

CBSH, GBPUA&T, Pantnagar, US Nagar