Does C4 photosynthesis occur in wheat seeds?

In our humble opinion, the debate about the conclusions drawn in a recent paper regarding the possibility of C4 photosynthesis being active in wheat seeds is one of the more interesting scientific debates raging at present.

The debate started with a paper published by Rangan et al last year which described findings they had made about differential expression of genes implicated in C4 photosynthesis within wheat seeds. These expression levels differed to the gene expression levels in other parts of the plant. We wrote an article describing the research here.

The response to that article (and our post) was that the evidence provided, although novel and interesting, wasn’t enough to justify the conclusion that C4 photosynthesis was in fact occurring within the wheat seed. Noting the criticisms, we wrote an updated piece covering two published responses to the Rangan article.

But the debate hasn’t stopped there…

Back and forth we go

The journal Plant Physiology published a series of letters between the researchers who published the initial research and two researchers with the differing opinion. The series of letters outlines the main points of difference between the propositions.

In defence of the C4 pathway conclusion

The series starts with a defence of the conclusions drawn from the research. Citing the earlier work of Bort et al (1995), a paper which described labeled carbon assimilation differences between leaves and seeds of wheat and barley and which found no significant difference between the location of the assimilated labeled carbon, the defenders argued that the research did not distinguish between carbon assimilated in the glumes covering the seed (the Bort et al paper) and carbon assimilated in the pericarp (the Rangan et al research).

glume-wheat

Dissection of Wheat Glume showing glume and grain.

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Figure from article showing cross-section of wheat seed, particularly the pericarp.

The distinction between the two asserted is that the Rangan et al research shows that pericarp, as opposed to the glume, demonstrates elevated C4 gene transcription.

Further, it is suggested that the carbon source supplying the pericarp is not from the capture of external CO2 but instead comes from the endosperm capturing respired carbon, the carbon being derived from bicarbonate in the developing seed tissue, and moving outwards to the pericarp for use in the asserted C4 pathway. For this reason it is suggested that the conclusions drawn by Bort et al, that there was no evidence for C4 photosynthesis in the ears of C3 cereals from their labeled-carbon pulse experiment, is a false basis to deny their conclusion as it failed to account for this inside-out carbon delivery.

To the contrary

The first point raised in contradiction to the Rangan et al research repeats the earlier criticism – that although the gene expression profiles reported in the disputed paper are novel, interesting and worthy of further research, they alone are not enough to justify the conclusions made. To be able to make such a conclusion, evidence demonstrating flux of the metabolites through the C4 pathway. Increased expression of the relevant genes is not enough to conclude that the pathway is operating in the seed particularly as all the genes involved in C4 photosynthesis are also expressed in C3 plants.

Although the evidence for the existence of the pathway in wheat seeds is scarce and contradictory, the defenders of the rebuttal suggest that the Bort et al article is evidence that the PEP carboxylase activity assists in intermediate reactions in metabolic pathways other than C4 photosynthesis.

Further, the evidence in the Rangan et al paper suggested that the expression of genes encoding the Rubsico enzyme was minimal. The contention is that, if the increased transcription of C4 genes is relied upon as evidence of increased activity within the pathway, reduced transcription of this vital photosynthetic enzyme must lead to the conclusion that the increase in PEP carboylase activity must be in aid of some pathway other than a photosynthesis pathway. Coupled with this, the low concentration of CO2 in the pericarp, where the Rubisco is located, is contrary to the high concentration of CO2 around Rubsico common in C4 tissues. Accordingly, expending energy to run a C4 pathway when the carbon would flow to Rubsico within the pericarp without the need for the pathway is contrary to expected energy conservation measures.

…and back again

In response to the criticism that evidence of flux through the pathway is lacking, the defenders of the Rangan et al paper suggest that such evidence has already been reported in a 1976 paper in the C3 intermediate barley and the relevant proteins isolated in research reported in 1986. It is argued that their research has showed that C4 specific versions of the genes were expressed in the seeds compared to the C3 versions expressed in the leaves.

In relation to the Rubisco levels, the Rubsico levels reported in the paper were for the whole seed, not just the pericarp and earlier work has shown that Rubsico is specifically expressed in the pericarp with very limited levels in the endosperm. As such, it is argued that the although the levels were rather limited in the seed as a whole, they are in fact present in high concentrations in the pericarp, the site where the C4 pathway is said to concentration CO2 levels.

Returning to the problem of the Bort et al paper, the researchers again suggest that the experiment conducted in that research could not have tested for the possibility of a C4 pathway supplying CO2 to the pericarp from within the seed, after which it would be utilised by Rubsico concentrated in the pericarp. Accordingly, they argue that the conclusions of that study fail to invalidate their conclusions.

As for the possibility that CO2 could more efficiently diffuse to the pericarp, it is contended that if that was the case it would not be concentrated enough at the site of the Rubisco and would therefore all too easily escape the seed. Their contention is that the presence of a C4 pathway in the seed is necessary for adequate CO2 concentration to the pericarp-located Rubisco.

…and one last response

The final of the letters concedes that the labeling of CO2 inside the endosperm may yield different results compared to the labeling study in the Bort et al paper, but cites the 1976 research as using isolated pericarps in their experiment and therefore doesn’t provide appropriate evidence support the C4 pathway inside the endosperm.

In relation to the diffusion versus C4 pathway dispute, it is suggested that any CO2 which is not passed from the endosperm to the pericarp through intermediates in the pathway would diffuse outwards following Fick’s law whether or not it moves through the tissue as malate. If it moved as malate, again the cost of such a pathway would be more than if it moved by simple diffusion.

Conclusion

The last paragraph of the final letter sums up the research as it stands and the debate about the possible conclusions that can be drawn. The research in the Rangan paper provides “an exiting new piece of the puzzle” in the quest to understand how wheat seeds increase carbon gain. It would seem that to put the debate to rest, the flux of the carbon through a C4 pathway must be demonstrated unequivocally along with measurement the location and activity of the required enzymes.

A passionate and reasoned debate over plant science which is wonderful to see.

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Speed Breeding

The generation time of an organism varies from organism to organism. One of the defining features of model species is their short generation times, allowing fast analysis of, for example, gene mutations on phenotype (think of mice, zebrafish and the fruit fly). The shorter the generation time, the greater the number of experiments and analysis which can be performed within a set period of time.

In the world of plant science, Arabidopsis thaliana is generally considered the model organism and many papers on genetics and gene technologies use A. thaliana as the subject plant from which study results are drawn.

But conclusions drawn from a model organism doesn’t always translate to similar results in another organism – think of promising medical studies in mice that haven’t had the same effect in humans. Accordingly, model organisms may not always be appropriate for use to draw conclusions about other organisms.

Such is the case with the translation of studies in A. thaliana in some important crops such as wheat and barley. The problem with studying genetic traits in such crops is that the generation time of the plant may be long, significantly impeding important research.

Introducing Speed Breeding

Given the problem with generation times, a small army of researchers led by Lee Hickey of the Queensland Alliance for Agriculture and Food Innovation (and recent recipient of the Queensland Young Tall Poppy Scientist of the Year – congratulations Lee) developed a new protocol for speed breeding and tested a number of important crop plants for their response to the new protocol.

The Protocol

The protocol is pretty simple. Instead of using a standard photoperiod used when growing plants under study, the researchers increased the day length to 22 hours and reduced the length of time the plant is in darkness to 2 hours.

In fact, the paper specifies three protocols that all use this photoperiod, each of the protocols having a slight variation in the other growth conditions including the third protocol which is meant to be a less expensive set up able to generate similar results to that found in laboratory conditions.

Testing the Protocol

The first testing of the protocol took place in Norwich, United Kingdom, and compared the growth rates of bread wheat, durum wheat, barley and a model grass Brachypodium distachyon when grown using the speed breeding protocol or grown under usual greenhouse conditions with no supplemental lighting or heating.

The crops grown in the modified setting consistently flowered earlier than the control plants, taking approximately half the time to begin flowering with B. distachyon taking an average of 26 days and wheat taking on average 37 to 39 days. A significant change in the amount of time required to reproduce a target number of generations.

To ensure that the faster flowering plants didn’t suffer from loss of seed viability, the researchers tested whether the speed-bred plants were able to generate the following generation of plants. The researchers quantified effects on three phentoypic variables related to viability, including:

  • wheat seed counts per spike – a decrease compared to control;
  • spikes per plant – healthy number in the speed-bred plants; and
  • viable seed production – no difference.

Moving back from Norwich to Queensland, the researchers tested a second but very similar protocol with spring wheat, barley, canola and chickpea cultivars. This protocol used high pressure sodium lamps to produce the 22-hour day for these plants and compared the effect of this protocol to the same plants grown in glass house with a 12 hour day/night cycle. This time, both treatment and control groups were subject to the same temperatures for the day and night portions.

Similar to the first test, the time to anthesis was significantly less under the speed breeding conditions, but this time the wheat plants produced more spikes, grain number didn’t show the same reduced effect as it did the first protocol and the flowering times of the different crop species were more uniform than in the control. The researchers point out that this last effect, crops flowering at the same time, is advantageous when genotype crossing is to be performed. Seed viability was retained even when harvested 14 days post-anthesis and subsequently subjected to cold treatment for 4 days, indicating that further time savings can be made in the production of generation times.

In canola and chickpea there was no significant difference in seed production between the treatment and control groups.

Speed Breeding

Figure 1 from article demonstrating the photoperiod and temperature ranges under the speed breeding (left) and control (right) conditions and the number of generations which can be produced in wheat, barley, chickpea and canola under each condition.

Overall, the number of generations that can be produced under speed breeding conditions in a year were:

  • wheat – 5.7 generations;
  • barley – 5.4 generations;
  • canola – 3.8 generations;
  • chickpea – 4.5 generations.

The possibility of using the speed breeding technique in conjunction with single seed descent breeding and research programs was the basis of a seed viability analysis in wheat and barley. Using 100-cell trays, seed viability was confirmed for both genotypes at 80% two weeks after anthesis and 100% at four week post-anthesis. Accordingly, single seed descent programs can also be used in conjunction with speed breeding.

A low-cost speed-breeding protocol was also tested. In this set-up, LEDs were used exclusively in conjunction with a conventional split-system air conditioner to create speed breeding conditions and garnered similar results.

What about effects other than generation times?

Although faster generation times are extremely useful, if the effect of the speed breeding is to alter the crops’ phenotype, research programs would be plagued by unwanted confounding factors and breeding programs would be rendered ineffective.

The research team therefore mutated the awn supressor B1 locus and the Reduced height (Rhd) genes in wheat and obtained the expected phenotypes under the faster generated crops.

Testing any variation in disease responses was performed by inoculating wheat spikes with the bacteria which causes fusarium head blight, one set of spikes belonging to a susceptible cultivar and the second belonging to a head blight-resistant cultivar. The speed-bred cultivars showed the expected resistant and susceptible phenotypes.

Finally, the use of plant breeding in genetic engineering programs was addressed by looking at transformation efficiency of barley seeds grown under the protocol and, again, found no difference when compared to conventionally grown transformed plants.

Conclusion

Hickey’s team has successfully tested a protocol that will greatly assist research efforts into these studied plants and whose results will likely be replicable in many other plants. They cite sunflower, pepper and radish as plants that have shown similar responses to being grown in extended daylight conditions.

The ability quickly test, for example, the effect of knocking out or over-expressing a particular gene will certainly lead to more efficient test results and, hopefully will be another stepping stone to overcoming some of the limits to efficient research we currently endure.

 

C4 Photosynthesis – Testing Candidate Maize Genes in Rice

It would be no surprise to regular readers of the Legume Laboratory that we hold significant interest in the possibility of engineering C4 photosynthesis into C3 crops. Projects such as the C4 Rice Project are dedicating their efforts to this goal and its promise of more efficient crop growth and improved food security in the face of a changing climate.

Scientific Reports recently published an article funded through the C4 Rice Project which was looking to take the next step in procuring this biological engineering feat.

Maize is an interesting plant as it contains the two forms of photosynthesis, utilising C4 photosynthesis in foliar leaves while husk leaves use C3 photosynthesis. Previous studies have compared the transcriptomes of the two types of maize leaves to identify differences in gene expression. Given both leaves contain the same genome, hidden within the 283 differences in gene expression should be those differences which convert a leaf from using C3 photosynthesis to using C4 photosynthesis.

Working from this base, the researchers selected 60 of these genes to analyse further. The choice to look at 60 genes instead of the 283 differently regulated genes identified was born of the lack of high throughput technology needed to test gene functions efficiently enough for all to be analysed. The 60 genes tested were selected as they were predominantly transcription factors or leucine rich repeat receptor like kinases. A maize ubiquitin promoter was ligated upstream of the each individual gene resulting in 60 gene constructs that were individually transformed into rice plants.

In order to identify whether the genes made any change to transgenic rice plants, the researchers compared the leaf anatomy of the transformed leaves to the wild-type leaves. Regression analysis of vein number and leaf width in rice plants found a linear relationship between the two (a wider leaf will have a greater number of veins proportional to the change in width when compared to a narrower leaf). Given the difference in cells surrounding veins in C4 photosynthetic plants compared to C3 plants, there is a difference in the space between veins in the two plant types. The hope was to identify changes in the ratio of vein number to leaf width in transformed plants, an indication of a possible change in the method of photosynthesis.

Results

The constitutive expression of 47 of the 60 genes made no observable difference to the ratio of veins to width of leaf normally seen in rice plants and the plants exhibited similar phenotypes to the wild type plants.

Three genes resulted in the transformed plantlets being unable to regenerate. The authors explain that rice tissue culture requires an excess of cytokinin to auxin in order for shoot growth to be promoted. Research into the three genes involved (ZmIDD16, ZmbHLH106 and ZmHCA2) revealed homologous genes in Arabidopsis that show evidence of effects on auxin biosynthesis. As a result, it was hypothesised that each of the three genes have resulted in an increase in auxin biosynthesis, throwing out the ratio of cytokinin to auxin needed for regeneration to occur, although further research was noted as necessary to confirm this hypothesis.

A different set of three genes disrupted shoot and root development, again believed to be due to alterations in the cytokinin/auxin ratio. The YUCCA gene seemingly caused the biosynthesis of excessive amounts of auxin, resulting in characteristic curled leaflets and hairy roots. A gene encoding a SMALL AUXIN UPREGULATED RNA (SAUR) disrupted the formation of lateral roots, with homologous genes demonstrating a disruption in normal auxin transport, reducing auxin-related root elongation and retarding initiation of lateral roots. The third gene (ZmSACL3) affected cytokinin, inhibiting its synthesis and resulting in similar phenotypes to that caused by the YUCCA gene due to the altered auxin/cytokinin ratio.

The overexpression of transcription factor R2R3 MYB caused intermediate veins to lose sclerenchyma cell connections to the abaxial epidermis. The loss of strengthening cells resulted in curled leaves. A different and novel gene resulted in the opposite effect on sclerenchyma formation, causing an increase in the proliferation of this cell type and resultant thicker walls than in wild type plants. Further, a couple of larger than usual bundle sheath cells around each vascular bundle formed. The result of the overexpression of this unnamed gene were infertile plants with short shoots and roots.

Figre 4 from C4 OX study

Figure 4 from article showing the effects of over expression of an as-yet unidentified gene. Figure b shows the reduced root growth while figure c shows the reduced shoot formation. Panel g shows normal sclerenchyma in wild type rice plants while panels h and l show the increased cell wall thickness described above.

ZmWRKY12, a gene which inhibited callus formation and therefore had to be inserted behind an inducible promoter to allow phenotypic analysis, caused dwarfing and reduced root growth in transgenic plants. The vein number to leaf width ratio was unaffected by the transgene but the normally lobed mesophyll cells in rice plants were lost in the transgenic lines. The orthologous gene in Arabidopsis is known to be involved in the formation of secondary cell walls, but these researchers have discovered that it also seems to play a role in cell lobing, a phenomenon for which the genetic basis was previously unknown.

Figure 5 from C4 OX study

Figure 5 from article showing the effects of ZmWRKY12 expressed in rice plants. The most interesting phenotype change was the change in mesophyll cell lobing from wild-type (panels d, f, and h) to the lack of lobing in the transgenic lines (panels e, g, and i).

Finally, three closely related Zinc finger nuclease genes showed normal vein spacing but caused spindly plants with drooping stems and leaves. It was theorised that the cause of the phenotype was due to alterations in the production gibberllic acid in the plants.

Conclusion

This paper is a great example of the incremental advancement of knowledge that builds upon earlier increments and is built upon by later increments. Those increments fill gaps in knowledge, or rule out particular cause/effect relationships, which help to direct and propel research towards its particular end (although, of course, research and enquiry need not always have a particular end to be directed at).

In the case of this research, the experiment resulted in some interesting and novel results, even though it didn’t yield any specific advance towards understanding the difference in gene regulation between the two forms of photosynthesis. That being said, we know that constitutive expression of any of 60 genes isn’t enough to trigger C4 photosynthesis. That leaves the remaining 223 genes to test, or perhaps a temporally regulated expression of one or more of the candidate genes, or some other combination of gene expression not yet contemplated.

The possible causes of C4 photosynthesis are numerous. This study also highlighted the deficiency in technology related to the efficient transformation, validation and phenotyping of transgenic plants. At present we lack the ability to quickly test over-expression of individual genes, and this is a relatively simple analysis to perform. The possibility that differential expression of a number of the genes being required to establish C4 photosynthesis requires significant advances in both wet lab and computation biology if we are to quickly make significant leaps in this type of research.

Great work by a great group of committed scientists.

Synbio in Space!

The challenges of surviving in space (with or without hostile enemies intent on dominating the universe) has made and will continue for a long time to make for a good sci-fi story. The resourcefulness required to survive in the vast emptiness between (and on) planets also forces us think deeply about better ways of propelling, feeding, housing and medicating ourselves. The cost of launching extra weight into space plus the limited lifespan of necessities such as food and medicine leaves us with complex questions about how to sustain ourselves as we look to explore more of our solar system and, hopefully one day, beyond it.

A 2015 review in the Journal of the Royal Society Interface considered a number of space missions and analysed how synthetic biology may assist those missions in the four resource areas mentioned above; propellant, food, habitat and medication. Of most interest, given the significance of the challenges presented, is a six-person, 916 day return voyage to Mars which would include 420 days of traveling there and back plus a 496 day stay on the red planet.

What are we going to have to do to be real-life Martians?

Food

Although one of the shorter sections of the paper, how we will advance food production being the focus of this blog means that we will put the section of food production in space up-front and centre.

Based on astronauts living on the International Space Station, living in space requires 1.83kg of food per crew member per day. As a result, the six person Mars mission requires around 10 tonnes of food that needs to be shipped in order to sustain a mission if all of it was shipped from Earth although more accurate assessments of food amounts required to be produced whilst on mission have a lower estimate of 4.5 to 5 tonnes.

So the ability to produce food using available elements and compounds whilst away from the Earth is a significant problem. And the most accessible food production option is to use photosynthesizing bacteria and plants. Producing food using bacteria has the most significant weight savings, with Spirulina (a nutritional biomass made from either Arthrospira platensis or Arthrospira maximum) being promoted in earlier papers as a suitable option to producing food in space compared to attempting to grow and harvest plants. According to those previous papers, it is possible to produce 5.271kg of Spirulina per day using three 2,000 litre bioreactors. This rate of production would sustain the crew for the entirety of their stay on Mars and for the return to Earth and save a significant amount of launch mass.

Spirulina

Spirulina powder.

So how can synthetic biology help if Spirulina is already producible in adequate amounts to sustain the majority of a Mars trip? If you can imagine eating the same substance every meal for 706 days straight, one problem is easy to identify. It is suggested in the paper that synbio efforts could be directed to diversifying the textures and the tastes produced by the bacteria so that a variety of ‘foods’ can be consumed by those on the Mars mission.

Increasing the rate that the bacteria is able to reproduce will also aid the weight reductions at lift off and increase the redundancy built into the rations of food for the trip. There are some reported increases in production rates reported in the paper, but, for example, whether increased photosynthetic efficiency can be engineered into the bacteria to increase production is a question common to current research efforts in earth-bound food production.

Finally, being able to increase the nutrient content in the food produced to aid general health and space-specific health problems will increase the chances of a successful mission.

Pharmaceuticals

The shelf-life of pharmaceuticals are shortened in space travel and at present the issues caused by this are solved by  sending more supply missions. Given the time and distance involved in a trip to Mars, such a solution has little practical use.

Synthetic biology has the ability to play a role in readily producing pharmaceutical substances during a mission to Mars. For example, acetaminophen (paracetamol) can be produced using a modified chromisate pathway in E. coli. Unfortunately, space travel doesn’t suit E.coli as the compounds it requires to survive are not readily available. But if an autotroph can be manipulated to contain the same pathway and produce the same substance, we may have a new way to produce the pharmaceutical with the restricted inputs. The cyanobacterium Synechocystis sp. PCC 6803 has been identified as a possible candidate that will survive on the inputs available in space and could host the appropriate pathway to produce acetaminophen.

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Figure 8 from article demonstrating the proposed pathway to create acetaminophen from Synechocystis sp. PCC 6803.

It can be imagined that with enough research we should be able to produce a range of pharmaceuticals or at least their components from the limited inputs available in space.

Fuel

The further we go and the more weight we carry, the greater the fuel required. Complicating the matter further, the more fuel we carry into space, the more fuel we require to lift the extra fuel off the ground. The problem of needing more fuel to lift even more fuel to be able to propel more weight into space is one that permeates through the problems of supplying enough food and equipment to astronauts traveling for long periods of time.

The possibility of producing fuel in space is limited, according to this paper, to using methanogens to create methane. The type of methanogen used in bioreactors effects the quality of methane gas created and the rate of production. Further, a number of methanogens are able to use the compounds, particularly CO2, created by the crew of a space mission.

Synthetic biology uses to address the problem of fuel production in space will likely focus on the production of methane by methanogens. Particularly, the ability to produce methane using the least amount of inputs and with the greatest nutrient recycling efficiencies will be key. Similarly, modification of cyanobacterial production of ethylene, an alternative propellant, for greater production efficiency is another possible use of synthetic biology to aid space flight.

Habitat Construction

Lastly, the ability of erect a habitat on Mars will require either the supply of material from Earth and/or the use of material available on Mars with equipment supplied from Earth. The proposed use of 3D printing to construct habitat allows the use of finer materials in an additive manufacturing process. But the possibility of biologically produced materials (biopolymers) provides a method of producing the base materials using inputs available away from the Earth and significantly reducing the launch mass of materials dedicated to habitat construction.

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Figure 7 from article demonstrating the shipping mass saved for habitat construction in missions to Mars (left) and the Moon (right).

Synthetic biology efforts focused on increasing the synthesis of biopolymer material per unit of bacteria will result in less equipment in the form of bioreactors being required to travel with the crew with the time for construction of the habitat being reduced as well.

Synbio in Space

At its most basic level, the quest to man a mission to Mars creates problems that must solved using the most efficient means possible. As a result, the use of synthetic biology to solve problems in the four areas listed above looks little further than what inputs are available in space and how they can be used to solve the problems spaceflight presents.

However, advances in modifying organisms to meet these challenges will likely lead to flow-on effects in the same areas here on a better-resourced planet. For example, should it be possible to create different textures and tastes in Spirulina in the confines of space then one would expect that it will be possible to use different inputs and different genetic circuits to create a variety of foods. Further, food produced should be more efficient, sustainable and economical than traditionally generated food.

Genetic Underpinnings of Heat Resistant Potatoes

Identifying the genetic basis of a particular trait is the building block on which molecular and transgenic breeding strategies build upon. How we connect the dots between a genotype and trait is the basis of a past post “How do we find genes related to traits? A review of bulked sample analysis”.

Despite only being behind rice and wheat as the most important crop worldwide, quantitative trait loci (QTLs) for heat tolerance in potatoes have not, until recently, been identified.

Potatoes are particularly sensitive to heat, with temperatures above the ideal maximum of 22°C resulting in significant decreases in growth and yield. The higher temperatures have a number of effects:

  1. Strongly suppresses tuberisation;
  2. Reduces the proportion of carbon assimilated for tuber starch;
  3. Reduces chlorophyll levels and CO2 fixation rates;
  4. Halts tuber dormancy resulting in early sprouting and secondary growths.

A recent study in the Plant Biotechnology Journal sought to identify QTLs for heat tolerance by comparing differences in gene expression and tuber yield between progeny of a biparental diploid potato (06H1) when grown at 22°C and 28°C.

Results

Temperature response analysis and gene identification

Single node cuttings were used as a method to quickly induce tuberisation of subject potato plants, allowing a faster assessment of tuber yield (see this article discussing its use – paywalled unfortunately). 170 genotypes from the 06H1 population were analysed using 6 nodal cuttings from each to assess tuber yield under 22°C and another 6 for each genotype assessed at 28°C. On average, tuber yield at the lower temperature (1.017 grams) was greater than for nodes grown at the higher temperature (0.285 grams). The difference in performance was used in combination with single nucleotide polymorphism (SNP) maps of the 06H1 genome to identify genetic markers which may be connected to the genes which alter expression rates at the two temperatures.

fresh weight tuber yield nodal cutting assay

Figure 1 from article. Comparison of tuber fresh weight yield when grown at 22°C and 28°C.

Statistical analysis of the differences in expression levels at each marker found 3 markers which may be connected to the difference in phenotype. Using the Potato Genome Browser the researchers browsed the list of genes connected to the markers for suitable gene candidates for further research. Of the three markers, one marker contained a possible gene candidate, that encoding Heat Shock Cognate 70, a gene that has been suggested as involved in the heat tolerance in cabbage and potato.

With a candidate gene identified, the gene was isolated at amplified for analysis of allelic differences within the gene, of which 4 were identified and named A1, A2, A3 and A4. Nodes of potatoes containing each the four alleles were assessed for tuber yield, with allele A2 having the greatest fresh weight at both 22°C and 28°C and combined A2A3 genotypes having the highest yield. Gene transcript levels were assessed in plants grown at both temperatures which showed that the HSc70 gene was upregulated in A2A3 plants but not in the A1A4 plants, while the HSc70 allele up-regulated in plants grown at the warmer temperature were all from the A2 allele.

Analysis of the amino acids of each of the alleles themselves found that the A2 and A3 proteins contained an additional C-terminal sequence of KIEEVD (thought to be involved in interactions with co-chaperones), while no other variations were unique in a single allele.

What was unique to the A2 allele was a difference in the promoter sequence where a run of ten TA repeats was found as opposed to a run of four TA repeats in the promoters of the other repeats. The researchers later hypothesize that the greater number of TA repeats may result in enhanced expression levels, a hypothesis based on previous research in yeast, although the specific effect of the repeat TA sequence is not known.

Confirming the effects of the HSc70 A2 allele

To test the A2 allele and its promoter sequence, the gene and upstream component was transferred into Nicotiana benthamiana plants and subjected to 45°C heat in a growth chamber. After 24 hours the expression of the HSc70 gene was 7 times greater than in the control plants and cell membrane injury in the transgenic plants were significantly lower.

The unique promoter was then targeted for further analysis. The A2 allele was put downstream of a series of promoters with TA repeats varying between four and ten times. After being exposed to the higher temperatures the level of HSc70 transcription was measured. Alleles with eight to ten TA repeats in the promoter showed statistically significant elevated transcript levels compared to alleles with four to six TA repeats in the promoter.

HSc70 expression levels

Figure 6 from article. a) HSc70 expression levels for promoters containing 4, 6, 8 and 10 TA repeats and b) cell membrane injury percentage for plants containing the same numbers of TA repeats.

A2 allele-containing plants with significantly increased expression under 28°C heat were selected for analysis under 40°C conditions for 24 hours in comparison to wild-type plants. Extracts of plants after 4 hours at this temperature showed HSc70 expression being enhanced by up to 50 times more than in control plants. However, after 24 hours the transcript levels fell back to that seen prior to being subjected to the extreme heat. The A2 allele-containing lines has significantly less cell membrane damage compared to the wild-type.

Finally, the ability of the A2 allele to protect tuber yield at elevated temperatures was tested. Using the same nodal cutting tuber yield assay as before, A2 allele containing plants were compared with wild Desiree potato plants when grown at 20°C showed little difference in tuber yield. However, at 28°C the Desiree plants reduced yield by 75% compared to yield at 22°C. By comparison, transgenic lines had significantly lower yield reductions, with some lines have twice the fresh and dry weight yield as the wild type.

Conclusion

The identification of the heat shock cognate protein 70 within the potato genome and the quantification of the effects of the specific A2 allele demonstrates the significance of quality genome research, genome annotation and the ability of powerful molecular tools to seek out expression variances that allow us to pinpoint the basis of variances in phenotype. Coupled with well prepared and executed experiments, the development of these tools will continue to enable us to extract information that will assist us to assist crops and food production.

Whether it is through transgenic technology or molecular breeding, the identification of genes such as the one discovered in this study will assist food production meet the challenges of a changing climate.

Identifying the specific allele of the HSc70 protein that contributes to its resistance to the negative effects of elevated temperature is surely a big step into future-proofing the security of this important crop.

Increasing Plant Pest Resistance with Bt + RNAi Pyramids

Lately we’ve written a few posts about how crop resistance may be increased in the future. Our last post covered the development of a material able to pass RNAi protection to crops, and the one prior to that covered the multitude of sustainable new methods that may play a role in disease management.

This month, the Plant Biotechnology Journal (which is open – thank you Wiley) published an article looking at a possible way of reducing the evolutionary pressure caused by transgenic crops with one specific method of protection (Bt cotton in this study) as well as improving protection against pests that have developed resistance.

Bt cotton, a transgenic crop which contains a gene for a toxin (Cry toxin) found in Bacillus thuringiensis, provides protection against a range of insects. Prior to this gene being inserted into cotton, corn and a number of other crops, the Baccilus thuringiensis bacteria would be sprayed onto the crops to provide the same protection.

As successful as Bt crops have been, the use of a singular method of pest control puts pressure on the pests to evolve a method of overcoming the effects of the control mechanism. In the case of Bt cotton crops, farmers have begun using lines of transgenic cotton containing a number of different Bt toxins designed to kill the same pest. However, these pyramids of multiple Bt toxin genes are not entirely effective due to the toxins interfering with each other or causing cross-resistance within the pests.

Pyramids of Bt toxins and RNAi

The research tested the possibility of constructing a cotton plant containing both a Bt toxin gene and one of two genes for a double stranded RNA aimed at interfering with two particular genes within the pest Helicoverpa armigera, a moth which develops by feeding on important crops such as cotton and corn.

To test whether the pyramids improved crop defence against H. armigera, cotton plants containing one of the two dsRNAs, the Bt toxin gene, a pyramid of Bt plus one of either of the RNAi constructs or a control plant were challenged with either a Bt susceptible or Bt resistant pest. Two essential genes which have previously been shown to effect pupation of H. armigera were the targets of the dsRNA constructs. The pest fed with an artificial diet containing either of the dsRNAs  was shown to result in an increase in pest mortality and those pests that did survive had a lower weight compared to those feeding on the control plant.

Cotton plants were then transformed via Agrobacterium tumefacnians-mediated transformation to create lines of cotton with either of the dsRNAs or a Green Fluorescence Protein as a control and then selfed the resulting lines to create lines homozygous for each of the genes. Resulting lines were shown through Southern blot analysis to contain only one of the inserted genes.

Susceptible pests fed either of the dsRNA transformed plants showed lower rates of transcription of the target genes.

Having created lines of cotton containing dsRNA able to effect pest growth, the researchers crossed the dsRNA lines with Bt lines in order create crops containing both traits and developed those lines into homozygous cotton plants with  the same number of copies of each of the genes.

Did the Pyramids make any difference?

Before testing the pyramided crops to compare their effect on Bt resistant pests to that of Bt-only crops, resistant H. armigera were fed Bt, transgenic GFP or non-transgenic cotton with no significant difference in their mortality or growth rates. Susceptible H. armigera pests fed the same diets showed the expected increased mortality when fed the Bt cotton diet.

When the resistant pests were fed the RNAi cotton crops, mortality rates were similar to that in susceptible pests, with both lines of pest having a mortality rate close to that of Bt susceptible pests when fed transgenic Bt plants. Of the two RNAi crops, there was no real difference in mortality rates and development between the two genes targeted by the RNAi constructs. Nor was there any significant difference between the effects of RNAi crops on resistant pest mortality and crops containing the Bt + RNAi construct, demonstrating that the pyramid did not have any effect on the pest save for the presence of the RNAi component of the pyramid.

Bt susceptible pests grown on the pyramid cotton crops did show an increase in mortality and days to development when compared to the RNAi crops alone. Using the index of multiplicative survival (“IMS” – comparing the mortality rates of the pyramid to the expected mortality rate of the pyramid which is calculated by multiplying the mortality rates of the pests when raised on crops containing only one or the other the pyramided genes) to determine whether the Bt and RNAi components were acting separately or in concert to cause the effect seen. Using this method of analysis it was thought that the two genes contained in the pyramid act independently against the susceptible pest.

Overall, the cotton crops containing the pyramids showed increased protection against both the susceptible and the resistant lines of pest.

bt and rnai pyramid figure 1

Figure 3b from article. Comparative mortality rates between susceptible and resistant H. armigera on wild type (W0), Green Fluorescence Protein transformed (GFP), Bt, two RNAi transformed crops (JHA and JHB) and Bt + RNAi pyramids. Astrix indicates statistically significant differences between the two lines of pest.

Just in case there is a possibility that Bt resistance resulted in a fitness cost to the H. armigera that may interfere with the analysis, resistant lines were fed on wild type cotton and the transgenic GFP cotton and their development rates monitored. Interestingly, resistance to Bt does come with a cost to development time, resistant pests having a 15 to 16% increase in development time compared to their susceptible cousins. Mortality between the two lines however did not show any difference.

What effect may use of the construct have in reducing resistance evolution?

Computer simulations were used to demonstrate what effect the use of pyramid cotton will have on resistance evolution in a number of scenarios with parameters taken from common growing conditions in northern China and varying levels of pest fitness cost of resistance and time for resistance development.

The amount of refuge land used in the scenarios had a significant impact on resistance development times. Using a refuge percentage of 50%, it was found that adding RNAi defence either in succession with Bt crops or in a pyramid crop increased the time to development of resistance against the defence when compared to Bt crops alone. Using pessimistic parameters for the development of resistance and fitness cost (faster evolution and little to no fitness cost associated with resistance development) and a refuge percentage of 50, it was demonstrated that the time to resistance increased by 5 years when RNAi was used in tandom with Bt crops while time resistance increased to 10 used when the two methods were used consecutively in a pyramid.

Bt plus RNAi Fig 2

Figure 5 from article. Simulations predicting years to resistance under a) realistic scenario, b) Optimistic scenario and c) Pessimistic Scenario with differing percentages of refuge area.

Conclusion

This may be the first time we have discussed a paper which experimented on something other than an important food crop. But Bt transformed food crops are in widespread use and the reliance on only one method of pest control results in the types of problems we are seeing today with the evolution of resistance. Therefore, developing the ability to provide more sustainable, longer term protection to crops could be fast-tracked using a technology like this where the gene targeted by the RNAi can be designed for a specific pest with minimal side effects on related species of insect.

The accuracy of the computer simulations is a little difficult to make out without a better knowledge of the underlying data but could be the basis of field tests and more sophisticated simulations.

The development of RNAi technology, from examples like this to the creation of crop protection technologies like BioClay, is impressive and seems likely to play significant role in the future protection of food production.

 

 

 

 

Using Clay Nanosheets to Give Plants Sustained RNAi-based Protection from Viruses

We have previously written about the possibility of using RNAi-based technologies to provide plants more sustainable and greater protection against viruses. RNAi, or RNA interference, is the protective process used in many eukaryotic cells against viruses which uses double stranded RNA (“dsRNA”) sequences complementary to that of a pathogen to silence the translation of that foreign RNA into proteins. It was recognised in a recent review article as one of the genetic technologies that could be used to provide sustainable crop protection in the future.

An article in January’s Nature Plants (sorry, the full-text article is behind a paywall) looked for a way to give RNAi the ability to withstand field conditions when topically applied to crop surfaces.

BioClay as a Delivery Mechanism

The researchers investigated the possibility of connecting the dsRNA to clay nanosheets (“LDH”) to form a substance, which the researchers called “BioClay”, that can be applied to crops and provide longer lasting protection than applying naked dsRNA.

BioClay nanosheets were created with an average diameter of 45nm. Loaded onto the nanosheets were dsRNA sequences complementary to segments of the pepper mild mottle virus (“PPMoV”) or the cucumber mosaic virus (“CMV”).

To check for successful loading, the dsRNA-LDH substances were subjected to electrophoresis. The fact that the dsRNA-LDH complexes didn’t migrate from the well at all was taken as evidence that the dsRNA had been successfully loaded onto the LDH. Sequences up to 1.8kbp were shown to be attachable to the LDH to form BioClay.

Transmission Electron Microscopy used to view the BioClay formed showed that the dsRNA chain is either adsorbed on the LDH surface or thread within a number of LDH particles.

The mechanism of delivering dsRNA to the plant relies on the LDH degrading into a residue when exposed to CO2 and moisture. This process and the ability for BioClay to delivery dsRNA to the plant surface was tested by suspending the BioClay on the leaves of tobacco plants and incubating under atmospheric-like conditions for 7 days. The residue left after 7 days showed decreases in aluminium and magnesium, the conclusion being drawn that the LDH had degraded. The process was also tested by incubating test plants with CMV-loaded BioClay and collecting the residue after a week, finding that the amount of loaded BioClay had been reduced, indicating that the BioClay is releasing the loaded dsRNA.

How topically applied dsRNA provides protection to the subject plant is still a matter for further research. To test whether the dsRNA was being taken up by the plant after being released from the degraded BioClay nanosheets, the researchers attached a Cy3 fluorophore to LDH alone, to a dsRNA alone and to a dsRNA-LDH compound and tested all three by applying them Arabidopsis thaliana. 48 hours after application, the leaves were examined with confocal microscopy to determine whether any fluorophores and therefore, presumably, either the LDH, dsRNA or dsRNA-LDH complexes, had been taken up by the plant. The researchers observed the fluorophore within xylem of the leaves treated with Cy3 attached to dsRNA and dsRNA-LDH complexes, but was not internalised in treatments that did not contain dsRNA. Further, in the dsRNA-Cy3-LDH treatment showed flurophore uptake in the spongy mesophyll.

Not only was the fluorophore shown to enter the plant when attached to dsRNA, but was also seen to be transported to new apical meristem leaves that had not been directly treated.

Further testing of the uptake of dsRNA was undertaken on transgenic Arabidopsis that contained a β-glucuronidase reporter, the aim being to test whether a dsRNA directed towards the reporter gene interfered with its expression. Interference was measured with a fluorometric assay and plants treated with dsRNA-GUS complexes (with or without LDH) showed decreased β-glucuronidase activity, indicating that RNA interference was being induced by the treatments.

Did the BioClay Persist Longer?

The first few tests showed the dsRNA was being taken up by the plants and causing RNAi, but does the use of the LDH nanosheets to deliver the dsRNA result in greater protection?

The researchers tested the usefulness of the LDH nanosheets in a number of ways. First, they again labeled the dsRNA complexes with Cy3 and applied them to Arabidopsis leaves. After leaving them on the leaves for 24 hours half of the leaves in each treatment group were rinsed and the fluorescence levels measured. Complexes that contained LDH displayed residual flourescence while non-LDH treatments had little-to-no fluorescence after rinsing.

The LDH complexes were next tested with an RNase to test the ability of the different complexes to withstand degradation. dsRNA and dsRNA-LDH were treated with RNase. After treating, the dsRNA was released from the LDH in that treatment group and the two sets of dsRNA subjected to northern blot analysis. It was shown that the dsRNA originally attached to LDH had been degraded to a lesser extent than the naked dsRNA.

bioclay-fig-3

Figure 3 from article – Figures a – d show the microscopy images of the 4 treatment types to detect remains of treatments after washing. Figure e is the northern blot result showing the levels of degradation of the dsRNA by RNase when attached to LDH or naked. Figure f compares the dsRNA present on leaves at different time points after being sprayed with either the naked dsRNA or the dsRNA-LDH complexes.

Similarly, when the dsRNA and dsRNA-LDH were applied to leaves and their continuing presence on the leaves detected after application, the non-LDH attached dsRNA was barely detected after 20 days while the LDH connect dsRNA was detected 30 days after the treatment.

Similar to the findings about the translocation of the dsRNA into untreated leaves, the researchers used northern blot analysis on purposefully untreated leaves to test for the presence of the dsRNA 20 days after the spray was applied, finding that where the dsRNA was attached to LDH, the dsRNA was still detectable.

But Does it Afford Protection Against Viruses?

Showing that the BioClay can caused directed RNAi in plants and persist longer on plants is all well and good, but it must also provide the plants with additional protection against viruses.

Using nectrotic lesions caused by CMV as a marker for virus resistance, the study showed a significant reduction in the number of lesions in leaves treated with dsRNA and BioClay. A similar test used a PMMoV challenge to test the number of lesions formed. The leaves were challenged with the virus 20 days after being treated with dsRNA complexes and the researchers found that only the BioClay complex provided significant protection at this time point, demonstrating a longer period of protection.

Similarly, when a double-antibody ELIZA was used to test for the presence of CMV in leaves 20-days post challenge, the percentage of leaves positive for CMV was significantly less in leaves treated with BioClay compared to those treated with LDH alone and the dsRNA alone.

bioclay-fig-4

Figure 4 from article. Fig 4a and 4b visualise the lesion number of lesions per leaf resulting from being challenged at different times after being sprayed with the various treatments. The most significant result was the significant reduction in lesion numbers in BioClay treated leaves when challenged 20 days after the treatment when compared to the number of lesions formed after the other treatments.

Protection afforded to the non-treated leaves was tested by taking leaves that emerged 20 days after treatment and using the same double-antibody ELIZA to detect the level of infection. The researchers found a reduced level of infection in the new leaves when the plant had been treated with BioClay.

Finally, the researchers used RNA-seq testing on leaves challenged with CMV, some of which had been treated with BioClay or dsRNA, seeking out viral RNA. Leaves treated with the dsRNA or BioClay showed virus specific RNA was at least 10 times less abundant than in non-treated leaves.

Conclusion

The researchers have demonstrated through a series of steps that LDH nanosheets have the ability to deliver dsRNA to plants, be subsequently taken up by the plant and seemingly distributed throughout the plant, to provide useful protection against viruses. Most importantly, the LDH nanosheets were demonstrated to provide better protection to the dsRNA from being washed off the plant or from being degraded.

Field trials are the next obvious steps for a technology that seemingly has the ability to provide significant protection in a sustainable manner. The ability of the BioClay to withstand field stress, UV radiation for example, would further cement this technology as one that may alter agricultural practices and improve food security. Whether the RNAi protection can be extended to other pests is even more exciting.

A great piece of research which gives hope that this biological phenomenon can be used to assist crop protection and food production.

 

Genetic Engineering and Sustainable Plant Disease Management

Some of the earliest and most prominent uses of genetic modification technology in crops have related to disease management. The insertion of a Bacillus thuringiensis gene into crops such as corn resulted in protection against damage caused by certain insects, eliminating the need for pesticides against those particular pests is one example. Another example, the ability of crops to thrive despite the application of glyphosate, was brought about by modifying crops so that the pathway affected by the chemical to cause plant death is cycled more regularly, helping the crop to survive.

A recent review penned by Paul Vincelli in the journal Sustainability overviewed the possible targets of genetic modification to increase pest control, how and what types of modifications can increase immunity and the possible risks that must be addressed if engineering resistance is to be sustainable.

How can genetic engineering enhance disease management?

Engineering Pathogen-Associated Molecular Patterns (PAMPs) recognition

A common feature of the immune system of many eukaryotes is the ability to recognise particular patterns on pathogens (“PAMPs”). The patterns are conserved across species of pathogens and, once recognised by immune cells as they survey the cells present in their host they trigger an immune response.

Whilst all plants will have the ability to recognise a range of PAMPs, they wont recognise all of them. Therefore, if one species of plant has developed the ability to recognise a particular pathogen and defend against it, identifying the requisite gene and transplanting it into another plant that is struggling to defend against the same pathogen will quickly enable it to muster its own immune defence against it.

Resistance Genes

Resistance genes, or ‘R genes’, allow a plant to overcome effector molecules used by pathogens to increase their chances of successfully invading a host. In a never-ending arms race, a pathogen will develop an effector molecule to enhance susceptibility of the host to infection, while the host will in develop the ability to recognise the effector and induce the immune reaction again. In  response, the pathogen may develop a new effector molecule, and the plant must again develop the ability to recognise the effector and respond when it is present.

The DNA encoding these new proteins developed by plants to detect new effectors are termed R genes, and the ongoing battle means that there are a multitude of R genes relating to a multitude of pathogens throughout the plant kingdom. Therefore, transferring R genes from a resistant plant to a susceptible plant will transfer resistance.

Transferring R genes can be done via conventional breeding, although some plants are easier and less time-consuming to cross-breed than others. Engineering the transfer of R genes will be quicker, more effective to use in difficult-to-cross crops and will enable more precise insertion of the genes, reducing the inheritance of unwanted genes along with the R genes.

Such a technique was used to transfer a gene from peppers which conferred resistance to bacterial leaf into tomato.

The obvious downside to conferring resistance this way is that the pathogen will again develop a new virulence method which will again need to be addressed, resulting in only a temporary resistance. However, helping crops quickly adapt to a new infection will help ensure short term yields while also allowing the engineering of specific resistance to specific pathogens as new effectors and R gene couples are discovered.

Giving Defence Responses a Boost

As well as increasing pathogen and effector recognition to allow immune responses to be initiated, increasing the size of those responses can also help combat specific pathogens.

An example provided in the paper is the use of a constitutive promoter from wheat to increase the expression of native immune gene which helped rice crops combat a number fungal pathogens including rice blast.

Changing DNA Sequences that Result in Increased Susceptibility

Some pathogens have developed the ability to exploit some required host protein to give itself a route of infection. The susceptibility genes encoding these proteins are problematic to deal with given the necessity of the gene product. However, modification to the gene, either natural or synthetic, which alters the protein enough to reduce their ability to be exploited by the pathogen but not so great as to render the protein useless in its required role, has been shown effective in increasing resistance.

Plants producing their own Antimicrobials

Like the Bt toxin producing corn crops that now protect themselves using the same chemistry used by conventional pesticides, crops producing their own antimicrobials provides a further potential basis for sustainable protection.

A suggested method of overcoming the serious threat to the citrus industry in Florida caused by citrus greening is to have the plant produce defensins from genes derived from spinach.

RNAi

The discovery that double-stranded RNA results in the silencing of genes with a complementary sequence has led the ability insert genes into an organism coding for a double-stranded RNA which will silence a specific gene or, of use to us in plant immunity, will silence genes within a parasite to reduce or remove their pathogenicity. Such was the case with the papaya ringspot virus in Hawaii, which was overcome by engineering the papaya to produce a dsRNA complementary to a coat protein gene of the virus, removing a virulence factor relied upon by the virus and saving the industry.

Removing Host Virulence Factors

Similar to removing or modifying susceptibility genes to remove a target route of infection used by pathogens, removing or modifying host virulence factors such as a particular protein which allows strong binding of the pathogen is a potential method of reducing infection rates.

Detoxifying the toxins

Many pathogens produce toxins which will attack particular targets of plant cells to allow easier invasion. Being able to render the toxin ineffective will in turn reduce infection rates of many pathogens, and having the plant produce these detoxifying compounds is a possible means of sustainably reducing crop destruction from disease.

Using CRISPR/Cas 9

CRISPR and its ability to make target endonuclease activity to specific parts of a DNA sequence has seemingly limitless uses, including as a disease management tool. By targeting an endonuclease to DNA inserted into a plant, such as replicating DNA of Geminiviruses, will disrupt the replication and infection rates of numerous pathogens and holds the possibility of being an adaptable method of crop production.

Balancing Crop Protection and Resistance Selective Pressure

Although there are number of methods of increasing resistance through targeted means, evolution doesn’t allow us to simply pick a single method to eliminate a pathogen; resistance is an ongoing concern.

However, if the ability to use multiple methods of increased pest management is developed along with the ability to rotate what methods are used, we may manage to increase our crop protection and reduce the selective pressure we put on pests.

Stacking multiple genes into plants is a promising method of achieving this end and Plant Artificial Chromosomes may be the scaffold on which these methods of disease management can be simultaneously used in crops.

Concerns using Genetic Engineering

The usual concerns are raised and, although noted as risks that must be managed where there is a dearth of evidence confirming the size of the risk, those concerns are largely dismissed.

The health risks of genetically engineered crops are an oft-raised topic in public forums, but the science on the lack of risk is largely settled.

Flow of recombinant DNA into related plant species is discussed but is again largely dismissed save where further research should be conducted to quantify such risks. Given the context of such genes being already present in wild-type plants and therefore already available for gene flow, the lack of evidence of microorganisms being transformed by transgenes plus the ability to design synthetic gene components so as to be not usable by prokaryotic microorganisms, the use of genetic modification use  has little chance of increasing the risk.

The control of genetic engineering by large companies and the promotion of monocultures are also raised as potential concerns related to using this technology in disease management programs. Again, the concerns raised fail to look at the current state of agriculture where large companies already hold the majority of patents for non-GMO technologies and where monoculture farming is a symptom of economic pressure unrelated to the use or disuse of genetic modification technology.

Conclusion

The review provides a great basis for further researching possible methods of sustainable disease management, pointing out the multiple paths that may be taken to research and develop protections against any particular plant pathogen.

The new technologies and any risks that come with them must be subject to rigorous risk analysis and the pros and cons weighed before implementation. Combining the use of multiple types of technology and managing the evolutionary pressure that could be caused if only one type of technology or only one target is used could create methods more sustainable and more targeted than those used today.

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.

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!