Activation and Repression of Transcription Factors via a Synthetic Construct in Arabidopsis thaliana

Being able to turn genes on and off as required is a significant goal in genetic manipulation. Synthetically constructed gene circuits which turn on and off in response to specific activators have previously been demonstrated and the ability knock out specific genes with such technology as CRISPR/Cas 9 has made gene manipulation easier and cheaper.

But the purpose of this particular article in the Plant Biolotechnology Journal (open-access = awesome) was focused on manipulating the mechanics of gene transcription repression to turn off gene repressors (and activate expression) and then reverse the process in subsequent generations as required.

Gene Transcription

Transcription factors, the proteins that form part of the machinery that transcribes DNA sequences into RNA, contain motifs that allow for them to be activated and deactivated. Regulation of gene transcription using the activation and deactivation of transcription factors is a necessity in all cells, allowing the expression of genes to be limited to particular moments within a cycle or at different stages of development.

The examples used in this study when demonstrating the novel bit of tech the researchers developed to force the repression and activation of different genes illustrate the need for this function well. One gene, MYB80, codes a transcription factor necessary for pollen development and the programmed cell death (PCD) of the tapetal in plant anthers. If allowed to transcribe one of its targeted genes unregulated, PCD occurs too early and the male flowers are rendered sterile, meaning the transcription must be regulated if the plant is to develop properly. This consequence was discovered in an earlier study when a mutant of the transcription factor was created which deleted part of the encoding gene which resulted in the loss of the regulatory motifs, likely meaning the the transcription factor was unhindered in transcribing its target genes.

Another transcription factor studied by the researchers was that encoded by WUSCHEL (WUS), required for the development of the organising centre beneath the stem cells in the meristem. Regulation of WUS activity is required to create distinct cell boundaries and a lack of regulation leads to disorganised growth and male sterility. Similar to MYB80, the transcription factor has a number of regulatory motifs at the C-terminal end of the protein which interact with corepressors.

Manipulating the function of transcription factors to regulate gene transcription is therefore another potential method of turning genes on and off to test the knock-on effects and potentially alter and improve plant characteristics.

Synthetically Regulating the Transcription Factors

How these regulatory motifs inhibit gene transcription, and how we might be able to cause the same inhibition, was the focus of this research. Using the MYB80 and WUS as targets, the researchers created what they dubbed “Conserved Sequence-guided Repressor Inhibition”, or “CoSRI”.

The name of the protein gives away how it works. This study and previous studies have found that within specific repressors are regions that are conserved across many species. Studies that have removed these conserved regions have resulted in the types of defects discussed above due to the unrestrained transcription of the genes targeted by the subject transcription factors. When the conserved sequence is reinstated, repression of transcription is reinstated and phenotypes return to match the wild-type plants.

cosri-fig-1a

Fig 1a from the article. The figure demonstrates how the CoSRI is targeted to the conserved section of the repressor gene and the section of the CoSRI interfering with the binding of the corepressor to the repressor.

So the researchers targeted these conserved sequences as being the sites to direct their newly constructed proteins, thereby building in some specificity to which repressors are targeted. Conjugated to this new protein is a repression-motif interacting region, a section of protein which interacts with the DNA binding section of the transcription factor, stopping the corepressors from interacting with the DNA binding domain of the transcript factor.

Testing the Constructs

Genes for the CoSRI proteins designed specific to the MYB80 and WUS transcription factors were transformed into Arabidopsis thaliana separately.

When the MYB80-specific CoSRI was introduced, the same silique abortion and male sterility were observed. Sterile lines analysed showed that the transcript levels of the CoSRI were at the same levels of greater than the levels of the endogenous transcription factors. Further, transcripts of the genes targeted by the endogenous genes were reduced in sterile lines.

Similarly, CoSRI genes specifically targeting the WUS transcription factor was transformed into A. thaliana, again resulting in altered phenotypes, specifically defective shoot meristems resulting in poor growth and failure of silique elongation leading to loss of fertility.

In short, the researchers had developed a synthetic construct that competed with corepressors for the ability to bind to the repression-binding motif of the transcription factor and blocked the active repression of gene transcription normally mediated by the transcription factors.

Restarting the Repression

Being able to stop the repression of gene transcription is great, but what about reversing it? The example of such a use given in the paper is forcing male sterility in, say, a hybrid plant species, but then being able to reverse the sterility later.

To achieve this, the researchers created what they termed a restorer construct which combined the first 188 amino acids at the N-terminus of the corepressor (the part required to repress transcription by the transcription factor) with a conserved region of the target transcript factor. The result is a protein that is directed to, in one example, the MYB80 transcription factor that carries with it the necessary part of the corepressor to repress the workings of the target transcription factor and restoring the wild-type phenotype.

cosri-fig-1b

Figure 1b from the article. The figure depicts the interaction of the CoSRI with the repressor to block corepressors from the repression binding site of the transcription factor, and the use of the restoring CoSRI to reverse the sterility caused by the first CoSRI.

The researchers tested whether the new construct interfered with the transcription levels of any other genes or the transcript levels of the MBY80 targets in wild-type A. thalianthat had not been previously transformed with a CoSRI, finding no interference.

In lines that had both the CoSRI and the restorer construct, sterility was reversed when the transcript levels of the restorer were similar to that of the CoSRI. Fertility was only partially restored or not restored at all when the restorer construct levels were significantly lower than their competition.

Conclusion

This new technique appears to provide an indirect and reversible method of altering the transcription of specific genes. Being able to stop and restart transcription factors for specific genes could be invaluable to plant (and other) research and the technique could find its way in the the toolbox of genetic engineering technology that is exploding at the moment.

The technology could also open up the possibility of being able to alter plant characteristics to suit seasonal requirements, particularly if we develop the ability to not just knockdown and re-initiate gene transcription but also to carefully calibrate gene transcript levels.

 

 

New Imaging Technique Identifies How Plant Tissue Growth is Orientated

Last October, the journal Developmental Cell published this article about tissue growth control in Arabidopsis thaliana, examining the effects of mutating a particular gene thought to influence plant growth.

The title of the article buries the lead a little though, as the method they developed to assess the effects of modifying this particular gene could potentially be put to good use in systems engineering studies.

Introduction

The article starts with the researchers noting the importance of the apical meristems in plants, being the source of stem cells which differentiate to allow plant growth at both the root and shoot ends of the plant. Root apical meristems are well studied and while the effects of different treatments on shoot apical meristems and consequent growth of the stem, leaves and flowers have been studied, how these tissues are initiated within the apical shoot apical meristem is still a bit of mystery.

The cause of the mystery is not a result of under-appreciation of the importance of this part of the plant, but due to the complexity of the structure and growth characteristics plus the difficulty of studying the inaccessible region of the plant in real time.

Plant Biology

Within the introduction the researchers underpin the importance of the shoot apical meristem with a description of its structure. It has distinct zones with distinct functions, including a peripheral zone from which come leaves and flowers and a central zone which replenishes the peripheral zone cells. Underlying these zones is the rib zone from which stem growth originates. The rib is named as it is due to it being made up of a cells which divide in a different orientation to the overlying layers.

fig-1-stem-apical-meristem

Fig 1 from article. Figure 1F (bottom left) shows the locations of the peripheral (PZ), central (CZ) and rib zones (RZ) within the stem apical meristem.

As can be made out from the coloured lines indicating the orientation of cell division in figure 1G above, cells within the peripheral and central zones divide horizontally (or perpendicular to the surface of the meristem), while cells in the rib zone divide so that the daughter cells are orientated vertically, causing upward stem growth.

These cell divisions are controlled and vary depending on the stage of plant growth. During the transition to the vegetative stage of growth the outer zones are active while the rib zone is restrained from cell division. During the flowering stage the rib zone is active and stem elongation rates are increased.

Therefore, knowing how the rates of cell division are controlled could lead to advances in crop growth and production, and the method of visualising the effects of manipulating genes could be invaluable to food production.

The 3D imaging method

Imaging the division of the cells within the shoot apical meristem in real time is something that has not previously been achieved but is a technical advance required to understand the underlying basis of plant growth. So, these clever researchers at the Norwich Research Park invented a way to do it.

Based on the fact that the cell wall is extended perpendicular to the mitotic spindle during cell division, the researchers thought it likely that the new wall will be thinner than the existing walls. To utilise this characteristic of cell wall extension and the information it provides about the orientation of cell division, the researchers cross-linked polysaccharides in the cell wall with fluorescing propidium iodide. The thinner, newer cell wall should fluoresce at a lower intensity compared the thicker, existing wall and therefore the orientation of cell division within the meristem can be discovered and imaged.

It was using this method that the great images shown in figure 1 were obtained.

Using the new imaging technique to test the effects of gene modifications

The researchers examined the role of REPLUMLESS (RPL), a gene which transcribes a transcription factor known to regulate stem growth. The method by which RPL controls stem elongation and the transitions between different growth stages is unknown however. Therefore, using the new imaging method, the study looked at the differences in cell division between a wild-type A. thaliana and an rpl mutant.

Imaging showed that the RZ of the wild-type was well-defined when the orientations of the cell divisions were analysed while cell divisions in the rpl mutant were less well organised, indicating that the differentiation between the different boundaries may be controlled by the gene.

Testing these findings, the researchers labeled cells with a Green Fluorescence Protein to track cells as they divided and the directions of tissue growth, finding that the outer regions of the shoot apical meristem grew laterally in relation to the stem. Conversely, the central region of the meristem divided and grew vertically and this was different between the wild-type and rpl mutant.

Delving further in the function of RPL, chromatin immunoprecipitation was used to detect regions of the genome bound by the transcription factor, finding that RPL interacts with a large number of genes that control stem development. Particularly, differential gene expression of a gene LSH4, a gene that controls organ boundary development, was detected.

Therefore, using their new technique, the researchers found the RPL affects the organised division of cells in the shoot apical meristem which in turn affects the control of growth during the various stages of the growth cycle. The result of mutating the RPL gene on plant growth can be seen in figure 6G and 6H from the article.

rpl-fig-6

Figure 6 from article. 6G is the wild-type and 6H is the rpl mutant, demonstrating the effect of the disorientated meristem divisions when RPL was not controlling stem elongation. Growth rates are shown in figure 6K while the orientation of cell divisions are demonstrated in the top left of the figure.

Conclusion

The researchers have developed a method of imaging cell divisions within the stem apical meristem which was used to study the method of growth control exerted by a gene known to play a role in stem morphogenesis.

Critically, the method described can be used by other researchers to research and test genetic modifications that could help increase crop productivity or adapt crops for better or more efficient growth in differing conditions.

 

 

Photoprotection and Crop Productivity

This recent research article in Science has received a decent amount of attention for good reason given the possible impact it could have on crop productivity through increasing photosynthetic efficiency. However, the approach to increasing efficiency in this paper varies considerably from the efforts to transport C4 photosynthesis into C3 crops more regularly seen.

Background

We have a good understanding of the working of photosynthesis and its use of photons and excited electrons to fix carbon. But built into this system is a protection mechanism that kicks in when the intensity of light is too great for the CO2 fixation capacity of the photosystems, a damaging state for the plant to be in. When the excitation energy is too great, the energy is dissipated as heat, a process called nonphotochemical quenching of chlorophyll fluorescence (NPQ).

At high light intensity, NPQ is a useful process. However, NPQ at light intensities lower than that which could cause damage to the delicate photosynthetic components results in a reduction in CO2 fixation. When a leaf goes from high light intensity to low light intensity, NPQ reduces accordingly. However, the transition of NPQ lags behind the transition of the leaf from high to low light intensity, resulting in a temporary reduction in CO2 fixation and, therefore, plant growth. Earlier research indicated that these losses were in the range of 7.5 to 30% of fixation rates.

These researchers therefore tested the possibility of increasing the speed with which the transition from photoprotection to full resumption of carbon fixation could occur.

The Biochemistry

NPQ results from a conformational change to the photosystem II antennae from an unquenched to quenched state, which results in the excess excitation energy being dissipated. NPQ levels correlate with the amount of photosystem II subunit S (PsbS) and the flux of xanthophyll cycle. While over-expression of PsbS in plants can enhance NPQ and photoprotection as well as increase the rate of change between quenched and unquenched states, overexpression can also reduce CO2 fixation rates when light intensity is at a level lower than that which could cause damage. Further, the lag between transition from quenched to unquenched states due to the effects of PsbS on NPQ is minimal (10 to 90 seconds) compared to the to the same transition when NPQ is triggered by zeaxanthin (10 to 15 minutes), a product of the xanthophyll cycle.

photoprotection

Figure 1 from article showing the factors affecting NPQ under different light intensity.

Therefore, the researchers looked at whether adjusting the xanthophyll cycle could assist the transition rate of NPQ. Specifically, they hypothesised whether accelerating the cycle and simultaneously increasing PsbS would result in a faster reduction of NPQ when leaves transition from high to low light intensity.

The Study

The researchers transformed tobacco plants with Arabidopsis-derived sequences for violaxanthin de-epoxidase, zeaxanthin epoxidase and PsbS and promoters for their expression in leaves. Transcript and protein levels of the three sequences were shown to be increased in the transformed plants compared to the wild type controls. Leaves of transformed and wild type plants were then subjected to fluctuating intensities of light. The NPQ relaxation rate due to the altered xanthophyll cycle increased significantly compared to the wild type, having an average relaxation time of 753 seconds compared to 2684 seconds in the wild type while relaxation due to the additional PsbS expression decreased from 21 to 15 seconds on average.

The recovery of CO2 assimilation was also analysed under the same fluctuating light. After transitioning from high to low light the CO2 assimilation decreased and was at a minimum 30 seconds after the transition before increasing again as the photoprotection relaxed. The rate of CO2 fixation increased faster in the transgenic lines with 9% higher fixation rates compared to the wild type tobacco plants.

Further testing the effects of the overexpressed genes on CO2 fixation, the researchers looked at the variation in CO2 fixation rates in response to variations in light. Two tests were performed: vary light intensity leaving enough time at each intensity to allow the fluorescence and gas exchange to reach a steady state, and varying light intensity every 4 minutes.

During the steady state experiment, the maximum CO2 fixation didn’t vary between the transgenic and wild type plants (both averaging 0.092 CO2 per absorbed photon), indicating that the overexpression didn’t effect the photosynthetic capacity generally as was seen in previous experiments.

npq

Figure 4 from article showing CO2 fixation per photon, quantum yield per photon and NPQ levels in the wild type and the three transgenic lines.

Under the alternating light experiment the CO2 fixed per absorbed photon was decreased compared to the steady state experiment but was greater in the transgenic lines (0.066CO2/photon) than the wild type (0.058CO2/photon), an 11.3% increase. Similar findings were observed in relation to quantum yield of whole-chain electron transport.

Plants grown under field conditions showed the same differences in fixation rates. Further, a randomised block design consisting of 12 blocks was used to test the agronomic performance of experimental and control lines, with 14 to 20% greater dry weight observed in the transgenic lines compared to the wild type with noted increases in leaf, stem and root weights and leaf area.

Finally, whether the transgenic lines may suffer from altered photoprotection under high intensity was tested in seedlings. After 2 hours being exposed to excessive light the photoprotection appeared to be similar or higher in transgenic lines.

Discussion

In the discussion section the researchers point out that under field conditions an individual chloroplast can be subjected to instantaneous and repetitive changes from high to low light conditions due to shading from other parts of the same plant or from nearby plants. The ability to reduce the response time of the photoprotective system will significantly assist crop productivity.

Further, stomatal conductance increases under high light conditions and remains so for minutes after transferring back to shade, resulting in excess water loss compared to when the leaves receive less than a harmful level of light. Speeding the relaxation of NPQ, the researchers point out, should also result in better water use efficiency.

Conclusion

This results of this research are really novel and demonstrate just how complex and evolved the photosynthesis machinery is. The xanthophyll cycle and PsbS are found in vascular plants, leaving open the possibility of transferring these faster transition rates to important crops.

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.