An Update to “Evidence that C4 Photosynthesis Already Exists in an Important C3 crop”

We have previously reported on an article in Nature Scientific Reports (“the Rangan paper”) that indicated the possibility that C4 photosynthesis was being carried out in the grain of wheat, an important C3 crop that belongs to a clade with no evidence that C4 photosynthesis has ever evolved within it. As C4 photosynthesis is more efficient in fixing CO2 than C3 photosynthesis, the possibility of the pathway being active in such an crop would give hope that it could be manipulated to improve its growth properties and that other important food crops.

Shortly after we posted our article it was pointed out that, although the evidence gathered in that paper are a useful analysis of gene transcript data, the presence of C4 gene transcripts should not amount to the conclusion that there is an active C4 biochemical pathway in the wheat grain. In the last week, two articles have appeared rebutting the conclusions of the paper.

“Seeds of C4 photosynthesis”

This article by Julian Hibberd and Robert Furbank in Nature Plants points out that although what is presented in the Rangan paper provides an interesting in-depth assessment of the transcripts derived from the wheat grain, the transcript data by itself doesn’t evidence a functioning pathway.

Whether the transcribed proteins are active and to what extent they are active cannot be evidenced solely from transcript data. If they are active, whether they are linked appropriately to form the C4 pathway for metabolite flux also needs to be evidenced if we are to accept the possibility of a functioning pathway.

The Rangan paper also cited differences in particular cell types within the pericarp of wheat grain and combine these differences with the abundance of C4 gene transcripts to hypothesize the possible existence of the two-celled C4 system. There were two problems with this hypothesis:

  1. No cell specific transcript evidence is given to support the assertion, let alone the protein and metabolite data. Although there are a number of different types of cells in the pericarp, the transcript analysis does not distinguish between these types of cells which would help confirm or deny a difference in transcript abundance between the types of cells; and
  2. Both the Hibberd and Furbank paper and the paper discussed below cited a protein labelling experiment which showed the PEPC protein (an important protein in the C4 pathway) is not found in the pericarp but rather the aleurone layer.

The paper concludes by saying that the transcript data isn’t enough to form a conclusion that engineering C4 photosynthesis in wheat will be any easier in wheat than in any other crop.

“Poor Evidence for C4 Photosynthesis in the Wheat Grain”

This article in Plant Physiology make the same criticisms about the lack of biochemical evidence supporting the claim of C4 photosynthesis in the wheat grain, the lack of evidence showing which cells within the grain are the origin of the transcripts obtained and that previous immunolabeling studies have shown PEPC being localised in the aleurone layer and endosperm, not the pericarp as suggested by Rangan et al.

A further criticism of the Rangan paper was the assertion made that the increase in PEPC and decrease in RuBisco transcripts is evidence of C4 photosynthesis. Again, the lack of biochemical evidence supporting an active pathway was noted. However, the authors also note that C4 photosynthesis still requires the use of RuBisco to assimilate the CO2 concentrated by PEPC and that an approximate ratio of one-to-one is required. Therefore, an over-abundance of PEPC compared to RuBisco doesn’t result in the increased photosynthetic efficiency we relate with C4 photosynthesis.

And a bit more…

We contacted Professor Robert Furbank, co-author of the Nature Plants letter, who was kind enough give us some insight into the issue, its historical context and how big a discovery it would be if C4 photosynthesis existed in wheat grain:

“It has been known since Tom ap Rees’ work in the 1970’s that a suite of C4 enzymes are present in C3 seeds but the flux is into amino acid synthesis and gluconeogenesis.

What I find interesting in the context of the Rangan paper is that the same mistakes were made which delayed the discovery of the C4 pathway by a decade. Careful measurements of photosynthetic flux which can separate a respiratory role of PEPC and other C4 enzymes from a photosynthetic one are required.  To ensure that RNAseq does not become the emperor’s new clothes, we must combine biochemistry, physiology and our new next gen sequencing tools to build a cogent and robust story.

I would be very surprised in C4 photosynthesis existed in grains.  The evolution of C4 was to concentrate CO2 around rubisco in a low CO2 atmosphere, which is certainly not the case in a developing cereal grain.  Even in the glumes, there is hard evidence that the initial evidence for C4 photosynthesis was flawed.”

Conclusion

The Hibberd and Furbank response to the Rangan paper acknowledges that the transcript analysis performed and reported on provide interesting evidence regarding differences in gene expression between different plant tissue, but warn that the gap between gene transcription and functioning protein pathways is something that must be bridged before the type of conclusion made by Rangan et al. can be made with confidence.

We must also admit that, in our post on the Rangan paper, our enthusiasm for the conclusions presented was greater than our critical analysis of evidence supporting it. We thank Steven Burgess and Julian Hibberd for their tweets pointing out issue with the conclusions drawn and Robert Furbank for taking the time to talk to us. In future, we will improve on our analysis of new articles and will seek out comments from relevant experts when significant claims are being made.

Quantifying synthetic gene transcription in plants

The article we write about today, “Quantitative Characterization of Genetic Parts and Circuits for Plant Synthetic Biology“, was published online in Nature Methods about a year ago. But its importance is such that we still thought it worth describing and to point out to readers other sites that have also provided excellent overviews of this paper (see The New Leaf, the GARNet Community Blog and Science Daily‘s write ups).

The Study

The researchers behind the study were looking to address one of the biggest problems in plant synthetic biology (and synthetic biology generally), being the ability to design gene circuits with a solid understanding of the rate of transcription of each of the genes within the circuit. Being able to predict and ‘tune’ the amount that a gene is transcribed will be a great step forward in allowing researchers and biological engineers to design and test circuits on computers, saving the time and expense of having to synthesise and test each component in the wet lab.

But biology, particularly multicellular biology, is messy and noisy and affected by so many different factors that we are unlikely to know and control each and every one of them. This study showed as much, but also showed that with careful testing we can develop the underlying knowledge required to develop general rules that will assist higher throughput development and research.

Using protoplasts derived from Arabidopsis cells to express the synthetic constructs, the researchers developed three small circuits. One circuit was an inducible promoter which, when induced by an externally applied inducer to begin transcribing the circuit, would result in the production of the protein firefly luciferase (F-luc), a fluorescent protein that can be detected. The level of fluorescence detected is a function of how much the gene is being transcribed. The second circuit was also controlled by an inducible promoter which, when induced by the same inducer, would result in the transcription of a protein with DNA-binding domains which would bind to specific DNA sequences, in turn repressing the transcription of those genes. The idea behind the two circuits using the same inducer is that the amount of fluorescence from the first would act as a proxy reporter for the amount of repressor being transcribed.

The third circuit synthesised contained a gene for Renilla luciferase (R-luc), another protein which fluoresces but does so at a shorter wavelength than the firefly luciferase and is therefore distinguishable. This gene was linked to a constitutive (constantly active) promoter that contained a number of DNA sequences that could be bound by the repressor. The repressible promoter contained DNA binding sites at different points to test the ability to fine tune repression rates with different repressor/binding site combinations.

nature-methods-fig-1-protoplasts-inducers

Figure 1 from article. (a) is the repressible promoter with DNA binding sites at different points. (b) is the three

The result were protoplasts that (at least theoretically) would fluoresce at the shorter, R-luc wavelength when there was no inducer added to the cell as the gene circuit containing the repressor would not have been induced for transcription. When the inducer is added and increased, the R-luc fluorescence would reduce proportional to the amount of inducer added (as more and more of the repressor is transcribed) and simultaneously the amount of F-luc florescence would increase.

In a less messy environment, the input-output response would predictable somewhat like an electronic circuit – an increase in the input would result in an equivalent (whether that be linear, exponential, logarithmic etc) change in the output. The aim of the research was to quantify the input-output ratio and see if a mathematical formula could be applied to allow predictions of input and outputs to be made.

The Results

But biology is messy and the input to output ratio over a number of reproductions varied considerably even though the generally expected increase in F-luc and reduction in R-luc as more inducer was added was observed.

So the researchers looked at where the variability could be coming from and how it may be accounted for so that predictable quantification of the transcription rates could be derived. To test this they examined the amount of F-luc fluorescence in the protoplasts when no inducer was added which theoretically should result in no fluorescence or, if induced by something other than the external inducer, should be fluorescing at the same level. What they found was that there was an unexpected variability in the fluorescence levels even when there was not external inducer added. When sorted by different batches of protoplasts which were prepared on different days, it was apparent that some variation in the preparation of each batch (which they weren’t able to explicitly identify) was causing a variation in the F-luc levels that was not controlled for (Figure 2(b) below).

noise-in-protoplast-data

Figure 2 from article showing noise in the protoplast fluorescence data grouped by batch and inducer type.

Given the noise wasn’t completely random, the researchers looked to mathematics to attempt a solution to their quest to help predict output levels despite the noise.

The Mathematics

It was assumed that the input to output relationship would function in accordance with the repressing Hill function. But analysing their data compared to that expected according to the Hill function showed that the output amount was the Hill function multiplied by some factor that was related to whatever was affecting each batch. What they hypothesised what that the ratio between average of fluorescence of all batches to that of each batch, with no inducer applied, should normalise the fluorescence levels and eliminate the batch effect observed. Calculating the normalisation factor in this way resulted in the reduction in noise between batches.

normalisation

Figure 4 from article showing the effect of normalising the input-output data using the calculated normalisation factor.

Reproducibility and Usability

To test the circuits and normalisation of output quantification in a monocot, sorghum protoplasts were transiently transformed with the same result – normalisation of the fluorescence values reduced or eliminated the batch effects.

Further testing was done to compare the use of transient expression in protoplasts to expression of the same circuits in stably transformed plants to determine whether one is reliably indicative of the other. Again, the purpose of confirming this comparison is to allow faster testing of circuits using transient expression with confidence that similar results will flow from the same circuits being installed stably into transgenic lines. Comparing the two in Arabidopsis protoplasts, the researchers found that luciferase expression was lower in the stably transformed protoplasts compared to the transient expression but the difference could again be normalised and fitted with the repressing Hill function.

Thus, it was suggested by the authors of the paper that by using these normalisation techniques, transient expression of gene constructs in protoplasts could be reliably used to predict the expression levels of the same constructs in stably transformed plant cells.

Conclusion

The paper addresses the importance of predictable, reproducible quantification of genetic parts in multicellular organisms that produce a lot of noise when trying to quantify results. It demonstrates the issue that will consistently arise as we attempt to address food security and environment concerns with technology aimed to produce larger yields with lower input and land usage. But it gives an insight into the ability we have to overcome the hurdle and begin designing and testing circuits with larger throughput and greater reproducibility.

And was clearly worth writing about again!

CRISPR Efficiency and Specificity in Arabidopsis

The CRISPR/Cas9 endonuclease system is likely to be the restriction enzyme currently in most widespread use. Its ease of manipulation to target different nucleotide sequences has made it valuable to systems, transgenic and synthetic biology, allowing researchers the ability to observe the effects of manipulating specific genes by knocking them out, down or up regulating them or for adding new genes into genomes.

Related to our recent post about how we identify genes related to traits, many traits that we research and possibly seek to modify, particularly in crop species, are usually controlled by more than one gene. Therefore, studying the effects of altering a particular phenotype characteristic requires the ability to target multiple genes in the one organism.

A recent PLoS One research article looked at the use of CRISPR/Cas9 to target multiple sites in one transformation event in Arabidopsis thaliana. Particularly, they looked at its efficiency transforming a number of genes and also assessed the rate of off-target modifications made.

The Study

The researchers targeted 7 genes from the GOLVEN (GLV/RGF/CLEL) gene family. The proteins produced by genes belonging to this family are important regulators of root stem cell development. In the discussion section of the paper the researchers also mention that the targeting of these genes combined with their selection of guide RNAs resulted in a diversity of DNA sequences being targeted and a significant number of identified potential off-target sites that may also be effected by editing. Therefore, they believe that their results targeting members of this family of genes will be representative of multi-site targeting in Arabidopsis generally.

For each of the seven genes two restriction sites were targeted. gRNA units targeting these sites were synthesised into clusters of 4 and the clusters containing all 14 of the gRNAs were then assembled and cloned into a  binary vector. Wild-type A. thaliana were transformed using Agrobacterium tumefacians via the floral dip method.

The Experiment

Six T1 plants generated from the transformed wild-type plants were screened for editing by sequencing one of the targeted genes (GLV1). Only one of the six plants screened showed a transformation at this location.

The leaves of 48 T2 plants generated from this one transformed T1 plant were sampled in a pool. The pooled DNA was resequenced and the reads mapped against the TAIR10 reference genome.

The criteria the researchers set in assessing whether the targeted genes had been edited was to see whether there were any insertion deletion (indel) events at or near three base pairs upstream of the PAM sequence, noting that while large deletions may not be mappable such large deletions are likely to be infrequent (based on their previous work and experience).

The Results

Editing Efficiency

Using the above criteria and assessing the pooled reads of each of the 14 targets, two of the targets showed no transformation (and from the table below, appear to avoided resequencing altogether. The researchers opined that in their experience such events occur in a similar frequency in single restriction events). Efficiency of editing in the 12 successfully transformed targets ranged from 33% to 92%.

journal-pone-0162169-t001

Table 1 from article.

Editing specificity

Being able to target multiple genes linked to a specific trait for mutation is great, but study results can be effected by the endonuclease causing mutations at other sites in the plant DNA. To assess the risk of this occurring the researchers used CRISPRP, a tool that can be used to find highly specific CRISPR sites and allows researchers to assess likely off-target sites within a genome for a particular gRNA. Assessing each gRNA with the tool and limiting the results to the top 20 possible off-target sites for each gRNA sequence, 178 possible off-target sites were identified and were covered in the sequencing data.

Analysing the near 43,500 reads that covered the identified potential off-target sites, the researchers found no indel events attributable to the endonuclease (presumably using the same criteria used to assess whether targeted sites were edited).

Thus, it appears that stacking gRNAs and targeting multiple genes at one time is still highly specific in Arabidopsis.

To check for any other possible off-target events that were not covered in the identified sequences the researchers altered the filters applied to the possible indel sets and obtained a further 4 potential sites that had an allele frequency with with the gRNA of over 3%. Screening these sites in both the wild and the transgenic lines showed a number indel events in both lines, suggesting, say the researchers, that the identified locations are susceptible to deletion events.

Finally, the researchers looked for possible translocations resulting from multiple editing events occurring at once. To do this, they looked for reads of targeted editing events that were contiguous with other editing events (see figure below) and found two independent events, demonstrating that such events can happen but at a very low frequency in Arabidopsis.

journal-pone-0162169-g002

Figure 2 from article. The sequences in part A of the figure are target sequences of three gRNA. Part B shows the sequence of the translocation of one of the sequences categorised by the concatenation of the target sequences.

The Discussion

The study suggests that using CRISPR to target multiple genes can result in a targeted and precise gene mutation. However, this is limited to Arabidopsis manipulation and these results do not necessarily extend to other plants. That said, Arabidopsis is widely used model plant for research and the findings here may assist researchers looking to manipulate arrays of genes at the same time.

The low transformation frequency of an admittedly small sample size is a little concerning when compared to high efficiencies of transformation generally seen when CRISPR is used. The researchers point out that this may be due to genes targeted being essential or late embryogensis related. But it would be great to see a replication of the study using other phenotype-related loci to confirm whether there is any transformation effect caused by the genes targeted.

Overall, the results of the study suggest that CRISPR/Cas9 editing continues to find new and useful application and may be of use to those tinkering with plant traits or synthesising new ones.

Speeding up the Recovery of Stable Transgenic Tomato lines

Improvements in the means and methods of studying transgenic crops are a science in their own right. Testing and analysing different model species for their ability to be reliably and efficiently transformed using a simple method, easily selected for and fast growing cycles has led to the adoption of such species as Arabidopsis thaliana as the plant of choice for this research.

A recent piece in Plant Cell, Tissue and Organ Culture (the article is behind a paywall, but here is a preprint) reports on an alteration to a standard regeneration medium used by the authors when regenerating transformed tomato lines. Noting that tomato plants had become commonly used in theirs and other labs for studies including biotic and abiotic stress resilience and with the successful transformation of the tomato genome by CRISPR/Cas9 likely to increase its importance, the authors performed a literature review looking for growth regulators that may reduce the time for regeneration of transgenic lines when combined with the zeatin growth regulator contained in their medium.

What they found was a number of articles that cited the use of indole-3-acetic acid (IAA) with positive regeneration effects but with no mention of its effect on regeneration time.

Therefore, the researchers tested the time for regeneration of tomato explants transformed with Agrobacterium tumefacians when grown on their standard medium compared to media containing IAA.

The experiment

The researchers took seeds of Solanum lycopersicum cultivar M82 and germinated them on a Murashige and Skoog based medium. Parts of the plants were cultivated a day prior to being transformed with A. tumefaciens that had a kanamycin-resistance plasmid inserted into it.

After transforming the explants they were plated on their standard selective media. After a week they transferred onto either the standard media as a control or on the same media containing either 0.01, 0.05, 0.1 or 0.5mg/L of IAA. After two weeks they were again transferred onto fresh media with the same concentrations of IAA.

The shoots were allowed to grow from the explants until they were 3mm tall and then they were transferred to a rooting media with either no added IAA or with 1mg/L IAA.

By the end of the experiment they had a total of 750 explants per IAA concentration.

The presence of the transferred DNA from the A. tumefaciens in the regenerated plants was confirmed by a β-glucuronidase histochemical assay and by PCR amplification of the selectable marker.

The results

The DNA form the A. tumefaciens was found stably inserted in the transgenic lines while control lines were missing the transgenic DNA.

Of all the tested media, those containing the IAA had a lowered transformation efficiency compared to the roughly 90% efficiency of the standard media described in the body of the article. Instead, the IAA media returned an efficiency of 48% and 54%.

Table 1 from article – Results for recovery of stable transgenic lines of Solanum lycopersicum cv M82 from Agrobacterium tumefaciens-infected cotyledon explants cultured on selective plant regeneration medium supplemented with different indole-3-acetic acid (IAA) concentrations

IAA (mg/l)

Total number explants

Total number rooted plants

Average transformation efficiency (±) SE*

Total time for recovery of transgenic lines (weeks)

0

750

660

88 ± 2.2

17

0.01

750

390

52 ± 1.0

15

0.05

750

375

50 ± 1.5

11

0.1

750

405

54 ± 1.2

11

0.5

750

360

48 ± 2.0

16

Transformation efficiency values shown are the average from 5 experiments ± the standard error (SE) calculated from 3 biological replicates *Average transformation efficiency was calculated as percent of stable transgenic lines recovered from the total number of cotyledon explants infected with Agrobacterium tumefaciens

As can be seen from the table above, the time for recovery of the transgenic lines were reduced by 6 weeks when grown on media containing 0.05 or 0.1 mg/L of IAA.

Given the results, the researchers now implement a new protocol for transforming and regenerating transgenic lines of tomato plants as per the figure below.

tomato-regeneration-standard

Figure 2 from article showing the revised method for trangenic tomato regeneration using media containing IAA.

Discussion

Six weeks saved might not seem like much of a win, but when multiple lines of stably transformed lines of plants are generated in a shorter period of time the following research can be conducted earlier and the knock-on effects of reproducing results, publishing papers or adjusting experiment conditions can be undertaken earlier.

Lots of little wins can lead to big results and hopefully this adjustment helps a number of researchers achieve results in shorter time-frames.

 

How do we find genes related to traits? A review of Bulked Sample Analysis

Here at Legume Laboratory we have written a number posts about research that has overexpressed a particular gene which has been linked with a particular trait such as resistance to drought or resistance to pest damage. The idea behind such research is usually to see whether an increase in the amount that gene is transcribed results in a linear change in the trait, evidencing  the control of that trait by the gene being overexpressed.

But how are those candidate genes initially identified?

The Plant Biotechnology Journal recently published a review piece on the process and current state of technology used in designing and sampling populations of plants to isolate particular differences in phenotype and identifying the genetic differences that cause the phenotypes of related plants to diverge. In particular, the review focuses on bulked sample analysis, a short cut to the more time and budget costly method of gene mapping all samples of a population. The end result can be the identification of one or a couple of genes controlling the trait of interest, but is usually the identification of a number of regions of the genome that are differentially expressed between the phenotypes, such regions called  Quantitative Trait Loci (or QTL).

 

How we find genes related to traits

Bulked segregants and variants

To discover which genes are involved in a particular trait within a certain species of plant we first need to pull together a population of the plant that shows variation in the trait of interest. There are two methods of creating this population, one of which uses a controlled population created from a specific breeding strategy (segregating population), the other creates a population from plants with phenotypic variation in the trait of interest which are derived from any population of that species ie the population isn’t raised through controlled breeding (a variant population). The idea behind both strategies is to obtain a population of plants which, in the next step, can by phenotyped for the trait of interest with particular attention paid to the most extreme variation ie plants showing significant drought tolerance versus plants most adversely affected by water deficit.

Sampling and phenotyping

After a method of developing a population has been chosen the plants are grown and a method established of scoring or classifying the different phenotypes being examined. An example may be the number of lesions formed on plant leaves as a result of a fungus or grain yield under varying water supplies. In the case of segregating populations the phenotyping may be carried through a number of generations of plants with individuals at the phenotypic extremes being selected for crossing to create the following generation, segregating the trait and, theoretically, the genetic underpinnings of the trait.

In establishing these populations care must be taken to ensure that only the trait of interest is being selected for.  The authors of the review emphasise the importance of reducing the signal-to-noise ratio and mention the development and implementation of precision phenotyping techniques and technology.

Where a particular type of stress is being selected for, the contrasting environments (one of high stress, one of lesser or absent that stress) need to be established and tested for concurrently.

Once the population for phenotyping has been developed under the required testing conditions, the plants are sampled. In most cases the sampling takes place by applying the phenotyping criteria to each plant, the end result being a spectrum of phenotypes that will usually distribute normally with the extreme phenotypes being at the tail ends of the distribution curve.

Obtaining results that are statistically significant rely on the population size and the number of plants at either end of the distribution curve. Variations in the sample sizes required depend heavily on factors such as the distance between genes related to the trait (and therefore the frequency of recombination), the number of genes related to the trait and effect size of a particular gene or genes on the trait.

bsa

Figure 2 from article. Four types of bulked sample analysis (BSA). (a) BSA for qualitative traits such as disease resistance with two distinct phenotypes (R, resistance; S, susceptible). (b) BSA for quantitative traits with normal distribution, among which samples from two tails (L: lower; U, upper) are selected and bulked. (c) BSA for multiple parallel bulks with individuals selected independently from the two tails of a normal distribution. (d) BSA with only one bulk available for the target trait, while the other tail was killed by lethal genes or due to severe stresses, when compared with individuals randomly selected from a control population under no stress with normal allele frequencies for the target trait; CK: plants from the control population, R: plants selected from the stressed environment.

For a population consisting of between 200 to 500 plants, the optimum tail size would be 20% to 30% of the population. As the total population being sampled from increased, the size of the tails to be selected will decrease. Large variations in phenotypes can reduce the sample sizes to 10% of a small total population (200 individuals), while QTLs associated with a small phenotype effect will require a much larger population (3000 to 5000 individuals) with each extreme phenotype being a selection of 100 plants from each tail.

Figure 2 above shows different methods of bulking samples for analysis. In the case of a trait that can be classified as a resistance or susceptible to a particular stress, the more resistant and susceptible individuals selected from the tails are used, while populations looking at a quantitative change in phenotype (such as grain weight) can be sampled from the extreme tails in one or multiple bulks from each end of the distribution. Where, for example, one treatment group fails to survive the treatment process leaving only one tail, the tail can be compared to a selection of control crops (figure 2d above).

Molecular analysis

Once the selected samples of the population are bulked they can analysed by various methods to detect differences or changes in genome, gene transcription or protein expression.

DNA analysis is the predominant form of molecular analysis. For many crops a set of DNA markers have been created from analysis of plant genome, based on such genetic landmarks as simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs) and PCR based markers. Using the markers as the basis for PCR amplification, as a most common example, differences between the genotypes of the two phenotype bulks can identified and mapped back to the genome. The result is demonstrated in Figure 2 above with its depiction of DNA bands or DNA expression levels and the connection between variation of plant phenotype and genotype.

DNA microarrays are increasingly being used in a similar manner for a faster and cheaper analysis.

Linkage maps can then be created which show how closely linked the DNA marker is to the gene or genes within the identified QTL.

Analysis of the transcribed DNA via RNA sequencing analysis methods can give a greater insight into the variation in gene transcription between phenotypes, although the effect of any non-transcribed DNA or levels of transcription cannot be assessed.

Protein analysis is a little more difficult to perform and borrows from immunology methods that use labeled antibodies to detect proteins within the bulked samples. However, mass-spectrometry  and Edman degradation are two methods that are being used to understand the primary sequence of proteins present within the samples with greater precision and without the need to have a range of antibodies that will detect the majority of proteins in the samples.

bsa-pipeline

Figure from article – the BSA pipeline, from population selection to application.

Applications of Bulk Sample Analysis and the Future

Bulk sample analysis, particularly bulked segregant analysis, is repeatedly used to detect the genetic underpinnings of particular traits and is widely used in agriculture-related science. When performed under tightly controlled conditions it assists researchers to isolate a particular trait from other variations in phenotype from which  base the identification of QTL can result.

And the depth of interrogation of the genetic basis of important traits is increasing as sequencing technology develops. The ability to effectively barcode segments of DNA before sequencing it in a large pool of DNA, allowing subsequent identification of the starting DNA, will hasten data gathering.

More important is the reducing cost of sequencing DNA. At the point where using markers and PCR or microarrays hardly differs in price to entire genome sequencing, the amount of data generated for analysis (and the number of computer programs developed to assist with the taks) will explode. It may be then that complex traits weakly controlled by a number of QTL will be identified with comparative ease.

The theoretical assistance these methods have for agriculture are the identification of genes that control particular traits which will then be used for screening and selecting crop breeding stocks. As the library of QTL increases, the ability to select seeds for particular conditions will assist food production levels particularly in the more trying of growing conditions.

 

Evidence that C4 Photosynthesis Already Exists in an Important C3 Crop

“The presence of all C4 specific genes in the genome confirms that natural selection may have already explored the options being considered by plant breeders.”

Rangan, P, Gurtado, A, & Henry, RJ, 2016. “New Evidence for Grain Specific C4 Photosynthesis in wheat”, Scientific Reports, vol 6. 31721; doi:10.1038/srep31721 (2016)

Edit: After publishing this article it has been helpfully pointed out in a series of tweets that the evidence of C4 photosynthesis in wheat obtained by these researchers doesn’t confirm that C4 does exist in the grain. Evidence of a functional metabolic C4 pathway is still required if the conclusion of the paper is to be accepted. Thank you to the people you helped to clarify. 

The above quote is buried under the sub-heading “varied expression pattern between wheat genomes” in this significant article published Nature’s Scientific Reports.

We have written a number of articles about C4 photosynthesis at Legume Laboratory, including;

Therefore, we wont rehash how significant an effect the ability to convert food crops from using C3 photosynthesis to the more efficient C4 photosynthesis and what we already know about it, save that the authors of this article suggest that a 30% increase in wheat yield is possible if the crop was converted to C4 photosynthesis due to the resultant improvements in water and nitrogen use efficiency.

But what these three researchers found could greatly assist the efforts of engineering or breeding crops with this important trait.

Taking 35 genotypes of wheat, they performed a trancriptome analysis on the developing grains at 14 days and 30 days after the anthesis (first opening of a flower bad, marking the start of the flowering period) and leaves to look at the variation in gene expression at the two points in time and in the different tissue. The transcripts, after being converted to cDNA and sequenced, were mapped back to the genome to ascertain the transcribing genes in the developing grains, specifically looking for genes related to C4 photosynthesis.

What was found

The authors describe three C4 photosynthesis subtypes based on particular enzymatic pathways, being the:

  1. NADP-dependent malic enzyme (“NADP-ME”);
  2. NAD-ME; and
  3. Phsophoenolpyruvate carboxylase (“PEPCK”).

These pathways, which differ from the RuBisCO common in C3 photosynthetic cells, are usually found in the Kranz Anatomy arrangement of cells found in most C4 plants, although single celled C4 photosynthesis has been found.

The transcriptome analysis performed focused on the searching for the presence of genes encoding the enzymes involved in these particular pathways given that this would be a good indicator of a different form of photosynthesis than the C3 photosynthesis taking place in the leaves of wheat crops.

Molecular evidence

What was found was the presence of all the genes (including typical isotypes) required for NAD-ME C4 photosynthesis in the caryopsis of the wheat grains, identifying their location in the genome and differences in expression rates at the different stages of development of the caryopsis and differences in expression in the caryopsis and the leaves.

  • Phosphoenolpyruvate carboxylase – was shown to be transcribed 125 times more compared to that in leaves of the crop. Further, working on the knowledge that RuBisCO transcripts were significantly reduced in C4 cells, they quantified RuBisCO transcription and found a 76 fold reduction in its expression in the caryopsis.
  • Aspartate aminotransferase – there were six copies of this gene in the cDNA libraries  but only two were the C4 types, both of which had an increase in transcription at 14 days post anthesis.
  • Malate dehydrogenase – two genes were found, with one of the two copies, the one thought to be involved in C4 photosynthesis, being differentially expressed in the caryopsis compared to leaves.
  • Malic enzyme coding gene – two copies were found, one of which (the mitochondrial targeted copy which supports C4 photosynthesis) was up-regulated in the caryopsis whilst the other was up-regulated in leaves.
  • Alanine transaminase – the gene involved in converting pyruvate to alanine in bundle sheath cells (and converting alanine to pyruvate in mesophyll cells) in NAD-ME photosynthesis reactions, was found in two copies. One of these copies was found to be expressed at a higher rate in the developing caryopsis.
  • Pyruvate, orthophosphate dikinase – comparing the expression of the gene between leaf and grains showed greater expression in the grain.

Of these six genes, phosphoenolpyruvate carboxylase (ppc) and alanine transaminase (gpt) require information about their sequence to determine whether the gene is involved in the C3 or C4 pathway (our article “Can We Synthetically Engineer C4 Photosynthesis” mentioned that many genes involved in C4 photosynthesis already exists in C3 crops but was used for other reactions).

The transcription analysis showed that gpt was expressed in similar amounts in both forms in all tissues of the crop and wasn’t analysed any further.

However, in relation to ppc, it was known that a substitution of an Arginine amino acid at position 884 in the C3 enzyme was well conserved, while C4 transcripts have been shown to contain Glycine at this position. Analysis of their transcripts showed neither Arginine or Glycine at this position, prompting further research into a number of related C4 crops and their alterations at this amino acid. What they found was that while C3 transcripts were conserved with Arginine at position 884, C4 crops related to wheat had been found with Serine, Glutamine, Glycine or Isoleuvine at the position. Therefore, the researchers suggested that transcripts with the conserved amino acid at this position were likely C3 genes while variability at this position, which may explain the improved efficiency of C4 photosynthesis, indicated a non-C3 photosynthetic use of the enzyme.

Cytological evidence

Not content with relying on the molecular evidence supporting a theory of C4 photosynthesis in the wheat grains, the researchers looked at previous research of physiological differences in cell composition of the grains. They drew on previous research that showed differences in the cross and tube cells the comprise the pericarp, particularly the differences in the number of chloroplast grana stacked in cross cells compared to tube cells. This division of labour in the photosynthesis process between the two cell types, similar to the division between bundle sheath and mesophyll cells in C4 plants, combined with the transcription of C4 specific genes targeted to this layer of the grain, led to the assertion of the researchers of the existence of C4 photosynthesis specifically in the grain compared to the C3 photosynthesis in the plant leaves.

c4-in-wheat

Figure 4 from Article showing differences in cross cells and tube cells in pericarp of wheat plants.

Evolutionary evidence

The sign of good research – triple checking the assertion that has been formulated. Not content with the molecular and cytological evidence, the researchers assessed the evolution of wheat within its genus to look at the evolutionary plausibility of the existence of C4 photosynthesis in the species.

This analysis contributed to the finding of the differing amino acids at position 884 of the ppc gene transcript described above. The lack of conservation at this amino acid position was extracted from the finding that wheat and related species held 5 copies of the ppc gene, one of which had a differing amino acid at this position when compared to the other 4 with the C3-conserved Arginine residue.

Evolutionarily, the researchers traced back the altered amino acid through the genus and suggested that the evolution of C4 photosynthesis has occurred on four separate occasions. Relating their finding to the types of photosynthesis found in the tribe of which wheat belongs (Triticeae), other members of the tribe contain the same number of ppc genes, all of which have one C4 copy of the gene, whilst the evolution of Brachypodium, which branched off from this line before the evolution of the species with C4 photosynthesis, remains a C3 plant with all five copies of the gene containing the conserved amino acid (see table 2 of the article).

How does this help us?

The conclusion suggests that the simple assignment of a category of photosynthesis to which a particular plant belongs to may not be correct or helpful to further research. As highlighted in this paper, a crop which has forever been thought of as a C3 crop due to an analysis of its leaves may miss the C4 photosynthesis busily converting light in another part of the plant.

What is also means is that the possibility of engineering C4 photosynthesis into C3 crops may be less a matter of forcing new circuitry into leaves and may instead be more targeted at finessing the already present C4 genes into being transcribed throughout the plant.

A very exciting find by these three researchers.

A New Way to Image Root Structure and Development

A cool new way to discern the effects of different growing conditions and/or genetic variations on a plant’s root structure has been discussed and tested in a recent article in Plant Methods.

This methods paper assesses the use of RhizoTubes, half metre high transparent tubes with a diameter of 18cm. Inside the tubes, pressed close to the outer surface, is a membrane consisting of an 18µm mesh which allows water, nutrients and microorganisms to pass through but stops soil or other growing media and plant roots from penetrating from alternative sides of the mesh. The result is an inner area holding the growing media for which the water content, nutrient content and microorganism content can be controlled and an outer area pressed against the outer tube in which the plant roots grow.

rhizotube-diagram

Figure from article. RhizoTube diagram showing inner and outer tubes and the membrane separating the root propagation area from the growing substrate.

By growing plants in the tubes the roots are forced to grow in a two dimensional matrix which is visible from the outside of the tube.

Plants can be grown in the RhizoTubes in greenhouse environments, allowing experimentation with temperature, humidity and the like and how these environments, interacting with differing genotypes or soil stress scenarios, affect root development and structure. Such insights may play a significant role in agriculture and our ability to predict what species or genetic modifications will be able to thrive in more difficult growing environments.

The RhizoTubes are mounted on roller conveyors and can be easily moved around. Most importantly, the tubes can be moved into an imaging area (named the RhizoCab) where a the root system is illuminated with red, green and blue wavelengths of light. The RhizoTube is rotated through 360 degrees and as it does so a high definition camera photographs the tube in one pixel wide increments. The result is amazing images like these:

rhizotubes

Figure from article. Examples of images (600) taken by RhizoCab of plant cultivated for 51 days (a), Pisum sativum plant (Cameor genotype) cultivated for 18 days with 10 meq soil mineral nitrogen (b) or without soil mineral nitrogen (c), Pisum sativum plant (Kayanne genotype) cultivated for 18 days without soil mineral nitrogen (d). Details of zone where either mycorhize can be seen or nodules (e) easily detected are indicated, with a resolution of 3600 (i.e. a pixel equals 7 µm)

As can be seen, the definition of the images of the root system are high enough for fine root structure and nodulation to be captured.

The aim of the device is to allow non-invasive assessment of root structures, avoiding the need to disturb the soil and roots of a plant under investigation as is required when such experiments are carried out in pots. The question the researchers were testing in this paper was whether the RhizoTubes distort normal growing patterns compared to growing the plants in a pot. Too much distortion and that results of experiments using the RhiztoTubes are unreliable.

Comparing RhizoTube growth with pot growth

Six experiments in total were performed with each experiment replicated in both RhizoTubes and pots. Set indicators were used in each experiment to compare the root growth between the two. The six experiments were:

  1. To test whether grapevine cuttings could survive and flourish when grown in RhizoTubes;
  2. To characterise root systems of pea roots and their nodulation between RhizoTubes and pots;
  3. To assess the effects of varying nitrogen availabilities on roots of different pea plants;
  4. To assess the differing responses to nitrogen availability on the crop Brassica napus and the weed Vulpia myuros;
  5. To assess what effect water deficit would have on pea roots and Medicago truncatula grown in the two containers; and
  6. To assess whether the presence of the membrane has any effect on the persistence of Psuedomonas fluorescences C7R12 on pea and wheat plants grown in RhizoTubes and whether the bacteria could be completely removed from the surface of the tubes after the experiment.

The results

In short, the differences between plants grown in the RhizoTubes to those grown in the pots were quite limited, but a couple of adjustments need to be made to ensure a like-for-like comparison in future experiments.

  1. The grapevine cuttings were able to develop in the RhizoTubes with comparable biomass ratios between the RhizoTube grown plantlets and those grown in pots.
  2. Pea root nodulation didn’t occur in either pots or the RhizoTubes when high levels of nitrogen were supplied and there was no difference in dry matter and root matter between the two containers. There was a difference in root length between the two containers, with the main roots from plants grown in the RhizoTube being 47% longer  and the number and length of first order roots emanating from the main root started decreasing further down the main root compared to that of the plants grown in pots. The second order root characteristics were comparable between the two containers.
  3. Adjusting nitrogen levels led to the nodulation of pea roots on both the main root and the lateral roots in both containers with Kayanne plants nodulating about 6cm higher in the RhizoTubes compared to the pots.
  4. Both the Brassica and Vuplia species tested developed a mess of thin, entangled roots which stopped the researchers from manually performing the root system architecture measurements. But it didn’t stop the RhizoCab taking some detailed pictures of the root structure. Although there were expected differences between the responses to nitrogen between the two plants, there was no significant difference between the growth of either plant in the differing containers.
  5. Performing a water stress experiment required a bit of fine tuning in the RhizoTube. Peas grown in RhizoTubes had lower (although only slightly lower) shoot and root growth although the same issue wasn’t found for Medicago. The authors hypothesise that although the RhizoTubes are suited to water stress testing of plants, fine tuning may be needed in how much and when water is provided in the tubes compared to the pots to ensure that a similar water level is attained. The same can also be said of nutrient provision in the tubes.
  6. The P. fluorescens was able to stably remain in the root system in the RhizoTubes and were the tubes were able to be disinfected of the bacteria at the end of the experiment.

The future

The RhizoTubesin combination with the RhizoCab imaging system appear to provide researchers the ability to monitor root differences between test subjects. As agricultural innovation continues to look for new ways to assist nutrient acquisition efficiency and adaptability to harsher growing environments, the ability to identify the phenotypic variation between genetically differing crops, and doing so with higher throughput than what exists at present, will certainly increase the rate of our knowledge gathering and application.

The main inhibitory issue remains the ability to phenotype roots of field plants, as opposed to pot plants. Perhaps the next iteration of this technology will be the capturing of 3D images in a similar device to that developed here.

Battling the Soybean Cyst Nematode

Soybeans are a great source of plant-based protein and produce considerable more protein per area of land than most other types of protein farming.

In the United States, one of the more damaging types of pest to soybean production is the soybean cyst nematode (SCN) and although there are some varieties of soybean that show some resistance to certain races of the nematode (currently the best known method of control), no variety is completely resistant and the number of known races of the SCNs mean that the threat of crop damage is constant and evolving.

With this in mind, a group of researchers from Tennessee (with funding from Tennessee Soybean Promotion Board) sought to build on previous research that had identified quantitative trait loci (QTL) associated with resistance to a variety of nematode races.

The article (from Plant Biotechnology Journal, which has been open-sourced by Wiley to its credit), cites previous research which identified a number of genes within the QTL that have been linked with resistance to some races of nematode. One in particular, the salicylic acid methyl transferase gene (GmSAMT1), had been previously studied by these researchers when they overexpressed the gene in soybean hairy roots and found that SCN race 2 development was significantly reduced. The GmSAMT1 gene plays a part in the production of salicylic acid (SA), a plant hormone that signals plant defence responses.

Developing this line of research further, the authors of the paper aimed to produce a number of generations of soybeans that overexpressed the GmSAMT1 gene to obtain further data on the possible correlation between the overexpressed gene and SCN resistance as well as looking at the effect of the overexpression on the plant’s agronomic traits.

The experiment

The researchers cloned the gene coupled with a promoter along with a herbicide resistance gene into a binary vector which was inserted into the soybean variety ‘Williams 82’ by Agrobacterium tumefacians. Transgenic lines confirmed to contain the DNA insert were self-pollinated to create a number of lines of third generation (T3) soybean plants. The level of expression of the GmSAMT1 gene was ascertained using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR – a method turning specific messenger RNA into DNA which can then be counted). A number of lines demonstrated higher than control levels of transcription  of the gene with one line of crops increasing GmSART1 transcription 226 fold compared to the control.

The transgenic lines were then inoculated with SCN. Lines were planted in two greenhouses, one of which was 5°C warmer than the other, and one lot planted in the field. The plants were left to grow for 35 days before being dug up and the number of cysts present were counted.

Transgenic leaf tissue taken after the 35 day inoculation period was used to perform a metabolite analysis of SA, benzoic acid (BA) and phenylalanine (PA) levels in the tissues for comparison to the control plant.

The results

As described above, the expression of the GmSAMT1 gene was ascertained for a number of lines of transgenic plants and the control plants. The expression of inserted gene was significantly higher in the transgenic lines (to varying degrees) compared to the control.

samt-transgenic-soybeans

Figure from article. Expression of the endogenous (figure a), inserted (figure b) and total expression (figure c) of the GmSAMT1 gene.

When the researchers looked at the infection of the plants with SCN they noted that the number of SCN eggs per cyst were not significantly different between the control and the transgenic plants, so they then counted the number of cysts on each plant to quantify the levels of infection. They found that there was a range of resistance in the transgenic lines with statistically significant reductions of infection with races 2, 3 and 5.

Correlating the total GmSAMT1 expression levels with the reduced levels of infection, a statistically significant negative correlation existed between gene expression and infection with SCN race 3, but not with the other races of nematode.

soybean-scn-infection-levels

Figure from article. SCN infection levels of races 2, 3 and 5 on control and transgenic soybean lines.

They then report their findings on the agronomic effect of the gene overexpression, finding that the transgenic lines were all significantly taller than the control soybean. They also found no significant difference in seed yield or in single seed weight. Interestingly, they found that the lines grown in the warmer greenhouse tended to have a higher seed weight per plant and in two transgenic lines the seed number per plant was higher than the control. The field experiment showed no significant differences in agronomic traits and there were no noticeable differences in plant phenotypes.

Finally, the researchers analysed the metabolites within the plant tissue using mass spectrometry. What they found was that SA and BA levels dropped in correlation with GmSAMT1 expression while PA levels increased in a statistically significant manner. They expressed an opinion on why this might be so in the discussion of their results.

The discussion

Many of the results speak for themselves so some of the discussion reiterated the correlations between transgenic plants containing the overexpressing GmSAMT1 gene and rate of infection of SCN found in the results.

The unexpected finding that the researchers point out was the non-linear correlation between overexpression and repression of different races of SCN. The transgenic line with the largest increase in gene expression had a significant correlation with reduced infection of SCN race three, while lines with lower expression of the gene had a higher resistance to races 2 and 5.

The researchers also point out the reduced BA and SA levels. They hypothesise that that a feedback look initiated by the reduced SA levels may have in turn reduced the levels of its precursor, BA, although why this occurred is not guessed at. PA, another important compound in SA biosynthesis was increased, was hypothesised to have been increased by the chorismate mutase enzyme, an enzyme that has previously been found to be secreted by numerous nematodes into plant tissue to manipulate the SA pathway. The precise explanation as to why PA levels affected by the nematode chorismate mutase enzyme in combination with the overexpressed gene would assist instead of hinder resistance isn’t given and is probably an area for future research.

In relation to plant growth and the noticeable increase in plant height and the increases in seed yield and number of seeds per plant in the warmer greenhouse, it is possible that increasing height is a strategy employed by the plant to overcome the deleterious effects of infection, while heat stress caused by the warmer greenhouse may induce a SA pathway response (previously been linked in other studies) which was increased in the case of the overexpressed GmSAMT1 lines.

What to take away

Taking one gene from QTL associated with a phenotypic trait is unlikely to yield broad spectrum resistance, and we don’t believe that the researchers expected such an outcome. What the research did do was correlate this particular gene and an increase in its expression with differing levels of resistance to a number SCN races, providing a useful building block to examine how genes within the other QTLs may help broaden the resistance. Further, it demonstrated that increasing resistance wouldn’t be done at the cost of the agronomic qualities of the plant.

What would have been interesting to see (although it may already be known – if it is, it doesn’t seem to be referred to in the paper), is what the metabolite levels were in a transgenic plant that hadn’t been infected with the SCN for comparison with infected transgenics and the control. From our reading of the methods, the mass spec analysis of metabolites was performed after the 35 day treatment with SCN and the data accumulated at that point. Given what we know about the secretion of enzymes into the plant by SCN and their effects on the SA pathway, identifying differences in metabolite levels between an infected and uninfected line may give a greater insight into any adapted response of SCN to the overexpressed gene.

Really promising research that gives some ideas of what further research is needed to gain the upper hand in this plant vs. nematode battle.

 

Can we synthetically engineer C4 photosynthesis?

“Photosynthesis as the engine for life on earth has high engineering potential, which has not yet been fully exploited…By step-wise identification of all the components needed for engineering, it will eventually become possible to employ this powerful machinery to increase yields for the future.”

Schuler, ML, Mantegazza, O & Weber, APM, 2016, ‘Engineering C4 photosynthesis into C3 chassis in the synthetic biology age’. The Plant Journal, vol. 87, pp. 62

These lines from the conclusion of the review we write about here are indicative of why so much effort is being put into understanding the more productive C4 photosynthetic system and working to increase important crop yields with it.

Schuler, Mantegazza and Weber’s article in the special issue of The Plant Journal on plant synthetic biology provides an excellent overview of the current status, significant hurdles and possible solutions to those problems of the current research aimed at bolstering rice yield by converting it from the common C3 photosynthesis system to the more efficient C4 system. We’ve previously written about C4 photosynthesis here and here.

C4 photosynthesis

C4 photosynthesis has evolved independently at least 66 times and is likely linked to a sudden drop in atmospheric CO2 levels sometime in the past. It is characterised by the concentration of CO2 around Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), the carbon-assimilating enzyme, reducing the competition that CO2 has with O2 to interact with the enzyme. More CO2 means greater growth and reduced photorespiration, an energy requiring process that is used to remove the O2 reaction products.

The concentration of CO2 in C4 photosynthesis is usually caused by a two-celled (but one-celled is possible) distribution of the process of fixing carbon and the process of reducing it. The two-celled system combines mesophyll (M) cells, which take up the CO2 from the leaf air space, and the bundle sheath (BS) cells, where the Rubisco enzymes reside, the final destination of CO2 for fixation and entry into the Calvin-Benson cycle. These two cells are arranged in concentric layers (called ‘Kranz Anatomy’) around leaf veins, maximising the contact between the two types of cells and increasing the transport of the molecules between them.

2000px-C4_photosynthesis_is_less_complicated.svg

M cells convert CO2 to bicarbonate and then into the 4 carbon compound oxaloacetate via an enzyme that doesn’t react with oxygen. The modified compound is then passed to the BS cells where it is reformed into CO2 and fixed by Rubisco to enter the Calvin-Benson cycle.

Basically, by assimilating CO2 away from Rubisco, the plant reduces the ability of Rubisco to interact with O2 and instead it is steadily fed with CO2 from the M cells.

Of course, this description of the process is simplified and although most of the process and main enzymes that carry out the process are known, there are still gaps in our knowledge.

Recent Advances

The gathering of increasing amounts of genomic, trascriptomic and metabolimic data continue to improve our knowledge of C4 photosynthesis, how it evolved and how we might transition C3 crops to use the more efficient carbon fixation method.

Important C4 crop species have had their genomes sequenced and quantitative analysis of transcriptomes have begun to unravel the mystery behind the genes upregulated and downregulated, and the stage of development that these regulatory differences occur, that lead the formation of the Kranz anatomy. What we are finding is that many of the genes involved in C4 photosynthesis exist in C3 plants but are differently regulated at early stages to differentiate the BS and M cells, enable high throughput of metabolites between the cells and to increase the size of vascular tissue to support the increased activity.

Engineering C4 photosynthesis

Our initial attempts to engineer C4 photosynthesis relied on over-expressing one or more enzymes in C3 plants. However, given the enzymes involved in the C4 system are used in the C3 system in multiple alternative pathways, the effects of over-expression were multiple, varied and didn’t have the desire result. The compartmentalisation of reactions, whether in the single or two-celled reactions that make up the distinctive photosystem, is complex.

The notion of being able to engineer C4 photosynthesis is comforted by a number of factors:

  1. The main enzymes are already present in C3 photosynthesis;
  2. Characteristics such as the passing of metabolites between cells is seen in C3 species such as tobacco plants; and
  3. Nature has done it herself in the past on multiple, independent occasions.

But the authors of the paper also note a number of engineering steps that need to be accomplished if we are re-enact evolution ourselves;

  1. Higher order veins need to be initiated in plants (it previously being shown that such physical properties were already evolved in plants that subsequently evolved the Kranz anatomy);
  2. The ratio of BS to M cells must be increased, ideally in a similar concentric organisation to Kranz anatomy;
  3. Enlarging and enriching BS cells with additional chloroplasts;
  4. Increasing the connection between M and BS cells;
  5. Engineering the different morphologies of the chloroplasts to mimic the morphologies of chloroplasts found in M and BS cells;
  6. Mirror the differing roles that M and BS cells take on in C4 photosynthesis so Rubisco reduction of CO2 occurs only in the BS cells with M cells feeding CO2 to the BS cells and excluding the oxidation of O2.

The tools we need

If we are to achieve success we still have some tools to develop and refine.

Chief among this list is a model plant that can be engineered and tested easily with speedy regeneration without requiring too much growing room. The authors point out that rice crops have some limitations in these criteria but identify Brachypodium distachyon as a model C3 plant with a small, annotated genome with quick flowering time, low growing space requirements and an efficient transformation protocol. A model such as this could hasten the engineering, testing and data gathering on conversion which can then be tested on important crop species.

A C4 model plant with similar characteristics is also required. Setaria viridis has previously been suggested as a possible model plant, as has the Fast Flowering Mini Maize.

The ability to drive and control expression of a transgene is also required. Cis-regulatory modules that promote gene expression are still under development in the wider plant synthetic biology area. This leaves a chasm between the tools we have to hand and the possibility that a large number of genes need to be differentially expressed in order to convert C3 photosynthesis to C4 photosynthesis.

Huge strides are being made with genetic manipulation, particularly with the discovery and modification of the CRISPR/Cas 9 system. But, according to the article, the maximum number of genes successfully introduced into a plant, at present, is 9. To induce C4 photosynthesis in a C3 plant, we may need the ability to stably transform a far larger number of genes plus regulatory elements, and do so without disrupting the remainder of the genome or the phenotype characteristics of our food crops.

Even when we do have these tools at the ready, we are still missing some vital information about the genes and regulatory elements that compose C4 photosynthesis. Increasing our knowledge of minutia of genetic composition and regulation of C4 systems compared to C3 systems is still a top priority. Identifying genera with the underlying predisposition that have allowed species within it to evolve from C3 to C4 for comparative analysis, particularly species displaying characteristics of a C3-C4 intermediate with sister taxa displaying C3 and C4 phenotypes, would be idyllic in assisting the study of the evolution. The authors highlight Morandia  and Parthenium generas as possible true intermediates between C3 and C4 plants. Programs such as the Grass Phylogeny Working Group and the 1KP (1000 plants) project will greatly assist identifying and genotyping suitable candidates for understanding the genetics behind enhancing crop photosynthesis.

And some suggested means of pushing the research…

It is great to see that not only have the authors elucidated quite extensively the current knowledge and gaps within the field of C4 photosynthesis engineering, but have also suggested a couple of ways of advancing the research.

The first idea they suggested is synthetically replicating a simplified C4 photosynthetic system using known genetic components. The system replicates the targeting of specific enzymes to create a two-celled photosynthesis construct, limiting Rubisco to the BS cells using RNAi to interfere with its transcription in M cells. The article highlights specific transporters that can be used to transport the metabolites between the two cells.

A second suggested idea is using brute force to direct a speedy evolution of a C3 or C3-C4 intermediate species into a C4 plant. Identifying the minimum genetic requirements of a C4 plant in a candidate crop would then be followed by the repetitive growth under the selective pressure of a low CO2 atmosphere. By repeating genomic and transcription analysis of the evolving plant (if successful), a ‘mud-map’ of the road from C3 to C4 plants can be generated and be of enormous use to research seeking to synthetically install the same machinery.

Conclusion

Although its behind a pay-wall, get your hands on this article. Whether it be for a background in C4 photosynthesis or as a springboard for your own research, it is an area of immense potential that should be worthy of an X prize.

Combined effect of Biochar and Arbuscular Mycorrhizal Fungi on Potato Growth in Conditions of Drought and Low Phosphorous Availability

Potatoes are delicious. They are also the fourth most cultivated food crop in the world, led only by wheat, rice and maize. Therefore, not only are the delicious, they are important to global agriculture and hunger efforts.

However, they are also sensitive to drought and phosphorous deficiency, both of which could cause significant problems with future production.

A paper recently published in the Journal of Agronomy and Crop Science contains some useful information just in the introduction regarding the positive effects of arbuscular mycorrhizal fungi (AMF) on plant growth, particularly that it has been shown in a previous study by the same authors that the negative effects of drought and low phosphorous availability on potato plants can be reduced by inoculating the seed and soil with AMF. However, the authors of the paper sought to test whether using a biochar amendment, which has also previously been shown to have some positive effects on potatoes grown under salinity stress, will confer any additional resistance to drought and phosphorous stress when combined with AMF. The hole in the research regarding biochar effects in this setting rests on the differing results from previous experiments regarding the usefulness of biochar amendment and the need for a better understanding of how biochar affects AMF. This research could help to decide whether crop management can be optimised through a combination of the two treatments.

Another interesting piece of prior knowledge that was used in the experiment was the effect of using alternating partial root-drying irrigation, when one half of a plant’s root system is watered completely while the other half receives little water, and which half of the root system receives which treatment is alternated. The result is that the water deficient half of the root system activates the production of the stress-induced hormone abscisic acid causing partial closure of the stomata, increasing water use efficiency. Although this also results in reduced phosphorous uptake and reduced plant growth and crop yield, the addition of AMF to the soil compensates for and reverses these negative effects. This is due to AMF acting symbiotically with the plant to effectively increase the plant’s root system and therefore increase its ability to acquire water and nutrients.

The experiment

The experiment tested 16 different treatments to understand the effect of biochar amendment on potato plants. The 16 treatments were made up of a combination of phosphorous fertilisation levels (P0 being reduced phosphorous addition, P1 being full fertilisation), irrigation amounts (FI being full irrigation, PRD being partial root-drying irrigation), AMF incorporation (M+ being AMF inoculation, M- being no inoculation) and biochar amendment (B+ and B-).

The biochar was created from pyrolyses of Birch wood at 500°C. The AMF species used was Rhizophagus irregularis. The experiment was carried out in a randomised pot experiment with the PRD treatments being performed by using plastic dividers in the pots that created a water-tight separation between the halves of the pot. One seed potato was added to each pot in a sandy loam soil with each treatment being performed in triplicate. Each treatment was applied for 30 days with the PRD being switched between pot halves of that treatment every 6 days. After 30 days the plants were harvested and plant total and root biomass, leaf area, phosphorous and nitrogen uptake, AMF colonisation of roots and water use efficiency were measured.

In a separate experiment, the researchers tested the ability of the biochar to adsorb mineral phosphorous and nitrogen in a water solution in order to understand the adsorption characteristics of biochar alone.

The results

The results section, given the number of treatments, reads a bit like a shopping list. Table 1 gives a decent overview of the statistically significant effects of each treatment.

Biochar and AMF table 1

Table 1 from article – Statistical significance of treatment effects on ten measured criteria.

The most significant results for purpose of understanding the effects of biochar application were:

1. Effects on plant growth and AMF root colonisation

The researchers found that, save for in the P0 FI M- treatment, the application of biochar had a negative effect on potato plant growth. Where other experiments had found that the addition of AMF to plants under phosphorous and water stress reversed the negative effects of those forms of stress, the addition of biochar reversed the positive AMF effects. While the highest biomass was recorded in the P1 FI M+ & B- treatment, the lowest biomass recorded was in P0 PRD B+ treatments. Biochar amendment had no effect on root biomass.

Further, it was observed that biochar amendment restricted young potato plants from growing and resulted in the death of some.

2. Soil water dynamic and water use efficiency

The data from the water use efficiency in treatments when biochar wasn’t applied demonstrated that water use efficiency was increased under PRD irrigation conditions compared to full irrigation, in line with earlier research.

When biochar was applied, the effect of the treatment on water use efficiency was linked with the change in biomass. Therefore, the negative effects of biochar that lowered biomass in turn lowered water use. Therefore, in and of itself, biochar didn’t demonstrate any appreciable effect on water use efficiency.

3. Soil pH, water soluble phosphorous and acid phosphomonoesterases activity

Biochar lowered pH only in the P1 FI M- treatment, had no significant effect on pH in the other treatments and no significant difference between the presence and absence of biomass was found in relation to water soluble phosphorous.

4. Plant phosphorous and nitrogen uptake

Biochar treatments resulted in the decrease of phosphorous uptake in plants. The only exception was in the P0 FI M- treatment (therefore, it only seemed to benefit the crops if there was low phosphorous, no AMF but full irrigation). Across the range of treatments it appears that all the good work performed by AMF in assisting plant phosphorous uptake is undone by biochar addition.

Further, biochar addition decreased total nitrogen uptake in all treatments (save for FI P0 M- and P1 FI M+) and the decreased nitrogen uptake was more pronounced under PRD irrigation.

P and N uptake biochar

Figure 5 from article – (a) Plant P uptake (mg plant−1) and (b) Plant N uptake (mg plant−1) as affected by P fertilization levels (P0: without P fertilizer, P1: with P fertilizer), inoculation treatments (M−: mycorrhiza free substrate, M+: Rhizophagus irregularis), irrigation treatments (FI: full irrigation and PRD: partial root-zone drying irrigation) and biochar treatments (B−: without biochar, B+: with biochar). Error bars indicate S.E. (n = 2–3). Different letters on top of columns are indicating significant differences (P < 0.05) between B− and B+ treatments within same irrigation, P fertilization and inoculation treatments.

5. Biochar adsorption of nitrogen and phosphorous in aqueous solution

Biochar didn’t show any adsorption of nitrogen and a 0.96% adsorption of phosphorous after 24 hours in an aqueous solution.

Pot experiment findings generally

In all, the researchers found that the change in total biomass of the potato plants was linearly related to the change in phosphorous uptake in the plants across each of the different treatment types but the change of nitrogen uptake, although less significantly effected by the treatments, had a stronger linear relationship with change in biomass.

Plant biomass biochar figure 1

Figure 1 (from article) (a) Total biomass of plant (g plant−1), (b) Leaf area (cm2 plant−1), (c) Root biomass (g plant−1) and (d) AMF root colonization (%) as affected by P fertilization levels (P0: without P fertilizer, P1: with P fertilizer), inoculation treatments (M−: mycorrhiza free substrate, M+: Rhizophagus irregularis), irrigation treatments (FI: full irrigation and PRD: partial root-zone drying irrigation) and biochar treatments (B−: without biochar, B+: with biochar). Error bars indicate S.E. (n = 2–3). Different letters on top of columns are indicating significant differences (P < 0.05) between B− and B+ treatments within same irrigation, P fertilization and inoculation treatments

Like we said, a lot of results, and many of the generalisations that could be made had one or more exceptions.

Discussion

From the data obtained the following general findings can be stated as:

  1. AMF is brilliant as it has the ability reverse the effects that drought and phosphorous deficiency has on phosphorous and nitrogen uptake and, by extension, plant biomass.
  2. The ability of AMF to work its magic in drought and low phosphorous conditions was impeded by the addition of biochar.
  3. Biochar did work to increase biomass in one scenario, when AMF was not present, phosphorous was low and there was full irrigation. Why this it so? We don’t know.

The effect of biochar cant be put down to its adsorption of phosphorous and has only minimal adsorption of nitrogen. So, why are we seeing these negative effects?

In relation to reduce biomass, the researchers hypothesised that the effect of biochar on the soil structure, particularly additional porosity, would alter water and nutrient retention rates in the soil, lowering accessibility to it for plants and AMF. However, unknown factors external to the experiment were also considered possible causes of the reduced biomass effect. How the altered water retention capacity of the biochar amended soil could cause this effect is difficult to pin down. The soil water content didn’t appear to be affected by the biochar amendment, nor was the pH particularly changed, both factors which can see reduced time for nitrogen and phosphorous to be available to the plants. However, biochar is hydrophobic which may cause water to permeate lower in the soil much more quickly, taking with it the nitrogen and making it more difficult for the plant to access.

But what about the effect of biochar on the AMF? Colonisation of plant roots didn’t seem to be affected by the biochar amendment, so there may be some effect of the biochar on the ability of the AMF’s nutrient gathering or its symbiosis with the plant. However, this has been left unanswered and further study is required on how the two interact.

What to do with this information?

It is rare (perhaps a first) that we write about a negative study (although no study except a poorly designed one is truly negative – we always learn something). But, whether you’re a spud farmer or just growing tubers in the backyard, this article teaches us that:

1.  AMF are important and can reverse the effects of reduced water and phosphorous content in the soil but adding biochar as well wont help.

2. If you have full irrigation but low phosphorous and no additional AMF or method to incorporate AMF into your crop, adding some biochar may help increase phosphorous uptake;

3. Biochar addition too early in the potato plant growth stages may delay growth and potentially kill your plant;

4. PRD combined with AMF is useful way (more practicable if growing potatoes in a pot) to reduce water use but not lose biomass;

5. If you are a research, there is a big gap in our knowledge – what effect does biochar have on AMF – does it affect AMF uptake of nutrients in the soil, does affect the symbiosis between the AMF and the plant roots, or something else altogether?