Getting to the root of the problem

30 September 2015 - By: Caroline Wood

Getting to the root of the problem


Ran Erel digging up a maize plant Photo: Ran Erel



By Caroline Wood

Perhaps one of the most pressing challenges facing mankind is achieving food security in a world faced with climate change, a rapidly growing global population and environmental degradation. All over the world, plant scientists are engaged in research that seeks to push crop production beyond current limits. One area that often receives little attention is how crop roots could be targeted to increase yields, but at SEB Prague 2015, strategies targeting root improvement was the focus of one of the major sessions, “Plant roots: new challenges in a changing world”. Here, we present a selection of the exciting ways in which researchers are truly ‘getting to the root of the problem’. 

A second “Green Revolution”

In the opening talk of the session, Jonathan Lynch (Penn State, USA and University of Nottingham) made the case for placing roots at the centre of a “Second Green Revolution”. An essential component of the first Green Revolution – artificial fertilisers – fuelled a system of agriculture which depended on intensive chemical inputs to improve yields. However, due to depleting resources and increasing degradation of the world’s soils, the crops of the future will have to be yield well without intensive fertilization; in other words, they must become more efficient. According to Jonathan, research in this area has been limited by approaches that focus on individual root traits rather than the integrated root phenotype. “Yield itself is not an individual trait but the product of many interacting elements”, he said. “If we can define these, we will have more powerful and precise breeding”. He illustrated this using the example of the Common Bean, Phaseolus vulgaris. The International Centre for Tropical Agriculture had worked for 20 years to breed a bean with highly efficient phosphorus uptake, without making much progress. But it appeared that beans with either a shallow root architecture or longer, denser root hairs had improved phosphorus efficiency.

Combining these traits in an additive fashion predicted a 150 percent greater phosphorus uptake. When this was actually done in the field however, phosphorus capture jumped 300 percent – twice that predicted from the additive benefits of the two traits. As Jonathan pointed out, this just shows that “we have to understand how root traits interact with each other in positive and negative ways”. 

The attention was then turned on maize – one of the world’s top three crops and a hungry consumer of nitrogen fertiliser. However, nitrogen is a highly mobile element and less than half of the applied fertiliser is thought to benefit the crop itself. Jonathan described how this is being combatted by plant breeders through a “steep, cheap and deep” strategy to promote the best root architecture for nitrogen acquisition. Computer modelling has suggested that plants have improved nitrogen uptake when they have fewer crown roots in the upper surface of the soil, so that each remaining crown root has more resources to grow into deeper soil layers where N is located. Field trials have since confirmed that plants whose roots plunge “steeply and deeply” into the soil acquire more nitrogen. It was also hypothesised that plants with roots made up of a greater proportion of aerenchyma (intercellular air spaces) would have an advantage.

Aerenchyma respires less than other tissues and requires less nitrogen and phosphorus, making it a “cheap” strategy to increase root growth. In the field, exploiting aerenchyma in this way was found to improve maize yields under low N conditions by up to 60%. The challenge now is to identify crop cultivars that combine these beneficial traits using a combination of basic “shovelomics” (“basically digging up plants and looking at them”) and the latest techniques such as Digital Imaging of Root Traits (DIRT). Using these strategies, Jonathan foresees a future where root systems are specifically tailored for different environments.

Know your soil

It is worth bearing in mind how the environment itself can shape root architecture. Most cereal production systems depend on unsustainably high inputs of phosphorous-enriched fertilisers, hence breeders are focusing their efforts on developing cultivars with enhanced phosphorous uptake. However, many candidates that show promise in the lab fail to perform under field conditions. “Because of the various factors that cannot be controlled in the field and the fact that it is nearly impossible to monitor root properties within the soil, most researchers grow plants in artificial substrates and under controlled conditions”, explained Ran Erel (INRA UMR EcoSols, France). “Hence, there is a need to validate the knowledge obtained in the lab under field conditions to identify the more important root traits for phosphorous acquisition efficiency”. 

At the meeting, Ran presented the results of a study investigating how critical root traits for phosphorous uptake differed in calcareous (alkaline) or neutral soil. This ambitious investigation was conducted on long-term experimental plots near Toulouse, France, treated with four different phosphorous regimes over 47 years. When phosphorous is limiting, there are two key strategies which plants can adopt. The first, “phosphorous foraging” requires investing in long, fine, dense roots to probe the soil. In the second, “phosphorous mining”, plants actively secrete enzymes from the roots or recruit microbes to help liberate recalcitrant phosphorous from the surrounding soil. A question relevant to plant breeders is whether one of these traits is consistently more successful or does the best strategy vary depending on the environmental conditions?

To test this, the researchers analysed root architecture and phosphorous acquisition in maize plants grown on the different plots. Curiously, under phosphorous limiting conditions, the root systems differed significantly between the calcareous and neutral soils. Although the total root biomass was similar between the two soil types, the plants grown on neutral soil had longer, finer roots with their level of phosphorous- acquisition positively correlated with root length. Conversely, on the alkaline soil the plants had wider, deeper roots and phosphorous availability was increased in the rhizosphere immediately surrounding each root. This indicates that under calcareous soil conditions, plants adopt a “mining” strategy to liberate phosphorous from the soil, but under neutral conditions they use a “foraging” strategy. “These results were very interesting and demonstrate that even relatively minor differences in soil property affect root traits for phosphorous acquisition”, Ran said.

It remains unknown through what mechanisms these differences influence root traits and whether the plants are responding to physical or chemical cues. Nevertheless, this study demonstrated the importance of putting new insights from the lab into their proper contexts.  “There clearly isn’t a “golden bullet” solution, where one trait will fit all situations” concluded Ran. “Instead, we need to match the right traits for the right environment”. 

Bring on the microbes!

There is much more to soil than its physical characteristics and Gabriele Berg (Graz University of Technology, Austria) eloquently highlighted the importance of the microbial communities to crop health. “Plant-associated microbes interact with the host plant in essential functions”, she said. “They can stimulate germination and growth, help plants fend off disease, promote stress resistance, and influence plant quality.” Whilst this has been known for many years, research in this area has been limited by the sheer challenge of characterizing the diversity of these rhizosphere communities. “In the past, we could analyse only a very small proportion - below 10% - of the microbial community”, explained Gabriele. “However, advancements in the “omics” technologies – including metagenomics and metatranscriptomics - have broken down these barriers and opened up new areas for investigation, including the contribution microbes make towards plant resistance to environmental stresses”.

Gabriele introduced us to the Arabidopsis Microbiome Project, a partnership between Graz University of Technology and MPI Golm in Germany. One area of investigation is assessing how microbial communities change during cold acclimation. In many areas, low temperatures are the key constraint on crop yields, however plants can increase their freezing tolerance through pre-exposure to non-lethal low temperatures (known as “cold acclimation”). Yet transcriptomic and metabolomic analyses have indicated that this phenomenon cannot be explained solely by changes that occur in the plant itself. Which raises the question – do microbial communities play a part in this?

In this project, the microbiomes of ten different Arabidopsis ecotypes, varying in their sensitivity/tolerance to cold acclimation were sequenced. It was found that each ecotype had a unique signature of microorgansisms, which changed markedly during acclimation. Certain genera, such as Sphingomonas and Buchnera were even unique to the most tolerant ecotypes. How exactly these organisms contribute to host plant resilience is currently a mystery and a fascinating avenue for new research.  It is hoped that this knowledge could be applied in the future to extend the cultivation range of temperature-sensitive crops. 

Greater use of “microbial networking” could also help us to protect our crops from deadly diseases, including the soil borne fungus Rhizoctonia solani. This is the causative agent of the devastating late sugar beet rot disease, which can cause up to 80% yield losses in some regions. Previous efforts to develop a biocontrol agent for coating seeds as protection against Rhizoctonia have had mixed results, with some cultivars responding well and others remaining highly susceptible to the pathogen. To investigate why this might be the case, Gabriele and her colleagues sampled the microbiome of different sugar beet cultivars grown in the field.

“We found that the cultivars showed clear groupings and had highly specific microbiome communities”, Gabriele said. It appears that the young plants recruit specific microorganisms during early development, presumably due to differences in the chemical compounds they release through root exudates. Furthermore, these specific microbiome complements have been shown to affect how well biocontrol agents establish on host roots. “I think we need a change in our relationship to sterility and microbes”, she continued. “It is clear that diversity avoids pathogen outbreaks and not vice versa”.

With technology placing the mysteries of microbiomes more closely within reach, it is certainly an exciting time to be involved. “Microbial ecology is undergoing a scientific revolution and it is very good to be able to take part in this”, Gabriele concluded. “It is so fascinating that we can open the black box and answer all these questions”. 

Finding the gatekeepers

Designing roots with the perfect architecture to scavenge water and nutrients may be one strategy, but what about the gateways that directly regulate uptake? One of the major routes by which water and nutrients gain entry into the plant is via aquaporins – integral membrane proteins that form water filled channels. These are typically encoded by large, multigene families (there are 35 in Arabidopsis alone) with many that are specifically expressed in the root. However this complexity has made it difficult to assess their potential contribution to drought-resistance.

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Photo: Francisco Perez-Alfocea



“Aquaporins can be regulated at both the level of transcription and activity and this is particularly important in situations where adjustment of water flow is physiologically critical”, said Zainab Abubakar (University of Aberdeen, UK). There are two contrasting theories regarding how exactly aquaporins function during drought stress. Certain studies suggest that aquaporin proteins are induced under dehydration to increase membrane permeability and promote water uptake from the soil. On the other hand, there is also evidence which implies that aquaporins are downregulated to reduce water lost from the root across the membrane. 

Recent work, however, has offered a clue into how aquaporins may be manipulated to improve performance under drought. In rice, an allele associated with drought-tolerance (called the Azucena allele) has been identified and this contains three candidate aquaporin genes belonging to the class of plasma membrane intrinsic proteins (PIPs).  To investigate the function of one of these, PIP2.1, Zainab used RNAinterference to knock down expression of the gene in rice seedlings. Once they were three weeks old, the plants were subjected to one of three watering treatments ranging from well-watered control conditions to severe drought. Root sections were then harvested and artificially shrunk by placing them in an osmotic solution. By measuring how much the root sections expanded when returned to water, the hydraulic conductance could be measured.  The results demonstrated that lower PIP2.1 expression significantly reduced hydraulic conductance, regardless of the watering regime, by as much as 25%. 

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Grafting a young tomato seedling. Photo: Michel E. Ghanem

However, despite the PIP2.1 knockdown lines performing well under well-watered conditions, they were more severely affected by drought with considerable reductions in shoot height and biomass. This indicates that aquaporins play a crucial role in obtaining moisture when it is limiting and that artificially raising their expression could be a strategy to breed drought-resistant cultivars. 

Curiously, low PIP2.1 expression also correlated with additional traits associated with drought resistance, including low stomatal conductance. “This is likely to be a mechanism to compensate for the reduction in hydraulic conductivity in the root (caused by reduced expression of PIP2.1) by limiting water loss by evaporation”, Zainab said. It appears that mechanisms for water balance show an interrelationship beyond our current understanding. 
 
“Aquaporins are proteins worthy of research”, Zainab concluded. “There is great potential value in identifying a gene that can be used to regulate the supply of water to the plant to either make it use water to grow faster or to conserve water and survive a long drought”. 

A load of hard graft

Francisco Perez-Alfocea (Centre for Applied Soil Science and Biology of the Segura, CEBAS-CSIC, Murcia) moved our attention from the latest, sophisticated technologies to an ancient, recently rediscovered agricultural practice - root grafting.  This technique involves splicing together the root and shoot sections of different cultivars in such a way that the vascular systems fuse to create a single, functioning plant. This can rapidly combine desirable root traits into high-yielding varieties, bypassing years of traditional plant breeding. Species of the Solanaceae (tomato, pepper, eggplant) and Cucurbitacea (melon, watermelon, zucchini, etc.) families are readily grafted, yet commercial grafting did not become common until the 20th century, after a scientific paper published in 1927 demonstrated that grafting watermelon  (Citrullus lanatus) onto pumpkin (Cucurbita moschata ) improved fruit yield and resistance to soil pathogens*. Since then, rootstocks have been used to improve water and nutrient uptake, increase disease resistance and extend growing seasons under extreme temperatures on an empirical basis. However, according to Francisco, we are currently only “scratching the surface” of this technique’s potential. 

Hence, in 2010, the EU-funded ROOTOPOWER project was launched to initiate an ambitious programme to screen an extensive population of different rootstocks, using tomato as a model crop. “Because tomatoes can be very easily grafted, this allows precise assessment of the effect of altering root traits on crop performance independently of shoot traits”, Francisco explained. “Tomatoes are also one of the most important vegetable crops with about 160 million tons produced globally in 2011, equivalent to 15% of total vegetable production”. Conventional tomato farms - large, heated greenhouses - are highly energy intensive and there is a real need to develop resilient varieties capable of being grown in the field. However, “traditional breeding for resistant varieties requires enormous efforts due to the multigenic nature of resistant or resource-efficient traits and the lack of genetic markers”, Francisco explained. “Many wild relatives show multiple abiotic or biotic stress resistances but have undesirable agronomic characteristics – but these can be circumvented if resistance can be rootstock-mediated”. 

So far, the project has established eight field sites across Europe to test the ability of the rootstocks to improve resistance to drought, hard soils and salinity, phosphorus, uptake of nitrogen and potassium and recruitment of beneficial microorganisms. Promising rootstocks with superior performance for these traits compared to existing elite commercial hybrids have been identified in populations derived from wild species such as Solanum pimpinellifolium, S. habrochaites and S. pennellii. As major outputs of those experiments, key quantitative-trait loci (QTLs) and candidate genes are being identified and validated, which could accelerate breeding programmes in tomato and other crops, including those that cannot be grafted easily. “In species that are not readily grafted, such as cereals, it can be difficult to separate traits mediated by the shoot or the root”, Francisco said. “However, using grafted plants allows us to more rapidly understand the genetic and physiological control of root-specific traits, facilitating the identification of causative genes for breeding programmes”. 


References:


Tateishi, K. (1927) Grafting watermelon on squash. Japan. J. Hort. 39:5–8.
Photo: Ran Erel
 
Category: Plant Biology
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Caroline Wood

Caroline Wood was the SEB’s 2014 science communication intern. Since then, Caroline has been a regular contributor the SEB, reporting on events and writing insightful features for our members.
Caroline has an undergraduate degree from Durham University in Cell Biology and is currently a PHD student at Sheffield University studying parasitic Striga weeds that infect food crops. You can read her blog here.