Opinion

The roots of a new

Griet Den Herder1,4, Gert Van Isterdael2,3, Tom Beeckman2,3 and Ive De Smet2,3

1 Genetics, Faculty of Biology, University of Munich (LMU), D-82152 Martinsried-Mu¨ nchen, Germany 2 Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Gent, Belgium 3 Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium 4 Current address: Ablynx nv, Technologiepark 21, 9052 Gent, Belgium

A significant increase in shoot biomass and seed yield production has often been overlooked. Nevertheless, has always been the dream of plant biologists who wish the root system is taking care of indispensable plant func- to dedicate their fundamental research to the benefit of tions such as uptake of nutrients and water, anchorage in mankind; the first green revolution about half a century the substrate and interaction with symbiotic organisms. ago represented a crucial step towards contemporary Consequently, root system development is central for the and the development of high-yield varieties plant to reach optimal growth and is sure to contribute to of cereal grains. Although there has been a steady rise in the levels of yield obtained in . Lately the impact of our food production from then onwards, the currently the ‘hidden half’ on plant growth has become apparent not applied technology and the available plants will not only in Arabidopsis (Arabidopsis thaliana), but also in be sufficient to feed the rapidly growing world popula- crops like wheat (Triticum aestivum), (Oryza sativa), tion. In this opinion article, we highlight several below- maize (Zea mays) and legumes, such as soybean (Glycine ground characteristics of plants such as root architec- max), barrel medic (Medicago truncatula), and Lotus japo- ture, nutrient uptake and nitrogen fixation as promising nicus [6–12]. Moreover, recent simulations suggested that features enabling a very much needed new green revo- changes in root architecture can strongly affect yield, lution. which might be sufficient to explain maize yield trends in the USA Corn Belt [13]. This indicates that root growth Rise of the hidden half and development might represent an underestimated and In 1798 Thomas Robert Malthus predicted in his An Essay not fully exploited area for strategies to enhance yield. on the Principle of Population that sooner or later a con- tinuously growing world population will be confronted with What are the major challenges? famine, disease and widespread mortality [1]. About two Due to climate change, plant roots and their habitats have hundred years later, the world is facing the major chal- a high risk of becoming subjected to unfavorable conditions lenge of providing for an ever growing world such as water scarceness, increasing ground water salini- population, while the agricultural area is shrinking [2].In ty, decline in soil nutrients and build up of soil pests. In the middle of the previous century, a Green Revolution addition the available arable land is becoming more sparse allowed food production to keep pace with worldwide pop- and precious due to erosion of hill-sides, soil degradation, ulation growth [3]. The International Food Policy Research landslides and the increasing demand for biofuels. The Institute has launched the 2020 Vision Initiative with the contemporary yields obtained by the classical use of water, primary goal to reach sustainable food security for all by fertilizers and pesticides have reached a maximum. 2020 and to cut by 50% the number of chronically under- Attempts to further boost yield by using more fertilizers nourished people on the planet by the year 2015 (http:// and/or pesticides are not feasible, not only because of a www.ifpri.org/book-753/ourwork/program/2020-vision- higher risk for public health and environmental problems, food-agriculture-and-environment). These deadlines are but also because of negative effects on yield [14]. For the approaching quickly, and we are far from reaching either root system to live up to the expectations as an important of these goals. In the immediate future, plant research will contributor to improved yield, a number of challenges will again be central in finding alternative crops or methods to need to be tackled (see also Box 1). cope with the threatening food shortage. In addition, next First, the conversion of unsuitable soil to arable land to finding the ideal food–population balance, improving will require agricultural techniques such as the improve- plant yield will also be vital for exploiting plants further ment of soil conditions through alternative fertilization. as a renewable energy source. Unfortunately, plant growth For instance, legume crop rotations as green manure have and productivity are greatly affected by environmental contributed considerably to the improvement of soil quality stresses such as drought, high salinity, nutrient-deficiency [15]. Second, and more critical, those soils will require and adverse temperatures. Due to climate changes these crops which are able to deal with the awkward edaphic challenges are currently becoming even more intensified. and climate conditions. We will need to improve root In the past, improvement of crops and agricultural architecture, nutrient uptake efficiency, nutrient storage techniques has mainly focused on increasing shoot biomass and root-to-shoot transport. Furthermore, roots need to and seed yield [4,5], and the relevance of the root system for fight off pathogens, prevent loss of soil through erosion and be able to resist the increasingly occurring unfavorable Corresponding author: De Smet, I. ([email protected]). conditions such as salt and drought. The past decade

600 1360-1385/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2010.08.009 Trends in Plant Science, November 2010, Vol. 15, No. 11 Opinion Trends in Plant Science Vol.15 No.11

Box 1. Hardest questions and a roadmap for root biology Box 2. The value of root research in the model plant Arabidopsis To obtain the necessary adaptations to root system architecture and symbiotic interactions, a couple of hurdles need to be taken first. A major challenge of studying model organisms, such as Arabi- How to translate the knowledge from conditioned, agar-grown dopsis, is transferring the knowledge and new tools to crop species model plants to soil-grown roots? How to translate the knowledge [4]. However, investigating root development in Arabidopsis has the from a simple Arabidopsis or legume model root system to a advantage that crucial genes can be more easily identified using complex crop? Next to answering these questions, as attempted genome-wide tools and integrated approaches, and quickly tested, throughout the main text, a number of key things that are needed upon which their function can be analyzed in crop species. For are: example, through a focused, cell-specific transcript profiling of early  A better understanding of root development and its interaction lateral root initiation we recently identified a ligand-receptor-like with the biotic and abiotic factors, as well as with the symbiotic kinase pathway centered around ACR4 that controls aspects of root organisms in the rhizosphere. branching in Arabidopsis [93]; and it will be interesting to analyze  More detailed analyses of the impact of root development on the role in root development of the maize ortholog CR4, the plant fitness, and consequently on the communication between founding member of this subset of receptor-like kinases [94]. root and shoot. However, because cereals display a more complex root branching  Integration of all aspects of root biology in the simple models that than Arabidopsis [8] it is clear that direct studies in maize, rice, exist for root meristem functioning and branching [87–91], and to barley (Hordeum vulgare subsp. vulgare L.), or the new cereal create models that implement the influence of root environmental model purple false brome (Brachypodium distachyon) play an conditions, such as nutritional sources and soil organism equally important role [95–98]. These and other species already associations [92]. revealed key regulators of (lateral) root development, before they were described in Arabidopsis [96,99–101]. In addition, Arabidopsis does not engage any root symbiosis, and therefore, these aspects provided an enormous leap forward with respect to our need to be addressed in model legume systems. Furthermore, more understanding of the physiological and molecular aspects research opportunities directly in the species of interest are arising, due to the increasing amount of knowledge and availability of of root development and branching in Arabidopsis [16] and genomic tools in crops, as well as the new sequencing technologies crops like maize and rice [6,8], as well as of root interac- which allow us to (re)sequence whole genomes and identify trait tions with pathogenic or beneficial organisms [17–19]. mutations in a straightforward manner in the crops itself. Never- Nevertheless, we are only at the verge of understanding theless, Arabidopsis will remain an excellent model system to study this complex process in its entirety. The remaining gaps in the basic elements of root architecture and adaptation to the environment, because of the vast knowledge on the developmental, our knowledge impede a speedy translation to agricultural physiological, and genomic level, and simplicity of the root system. applications and need to be filled by well thought-through research. Third, a major challenge will be to connect and combine those various genes and pathways that, individu- and increased root surface via root hairs have been studied ally, make a small contribution to a comprehensive net- in detail in various plant species [9,10,16,17,20–29]. Cur- work. Integrated approaches will be needed to identify rently, the most investigated regulator of root growth and central regulators of nodes where genetic, developmental branching is the plant hormone auxin. While increased and physiological pathways interact. levels of auxin clearly result in more lateral roots, it also leads to an impeded growth of the main root, hence giving Can we tackle the emerging problems? rise to a highly branched but superficial root system Yes, we can. Starting with fundamental analyses in the [30,31]. On the other hand, loss of either auxin response model plant Arabidopsis as well as model legumes and or auxin accumulation causes a dramatic reduction of root certain crop species, such as maize, rice and soybean it branching [30,32,33], and consequently a lack of root should be possible to design plants that fulfill the require- branching results in poor development of the shoot [34]. ments to contribute to an increased and more efficient root From these observations it is clear that direct interference system (see also Boxes 1 and 2). Here, we will summarize with auxin levels or auxin response mechanisms is not the current status of our knowledge – focusing on improv- adequate for a refined modulation of root growth and more ing root architecture and surface, and on nutrient uptake sophisticated approaches will be required. Next to auxin, and fixation – and point out what needs to be changed, several attempts focused directly on increased cell division added or should receive attention to achieve the set goals. to improve the root system, but it is now apparent that having more cells does not necessarily mean generating Root growth, branching and surface more lateral roots or a longer main root [32,35,36]. How- It is easy to think of a root system with a fast growing main ever, it is promising that more branching with only mild root, more lateral branches and/or higher number of root negative consequences for main root growth can be hairs as more efficient to take up water and nutrients, to fix achieved through combining an intermediate increase of fertile soil and to prevent soil degradation (Figure 1). auxin levels and increased cell cycle activity [32]. In addi- However, there are certain constraints, for example the tion, a fine-tuned control of cytokinin catabolism provides right balance between root biomass and the sources allo- another means to influence root branching and mass in cated to the root system has to be achieved in order not to both Arabidopsis and crops [37,38]. Since most likely a jeopardize shoot growth [10]. On the other hand, even general integrator of all signaling pathways at all devel- small changes in root angle affect root system architecture opmental stages does not exist, it will require a balanced and associated water capture, which can have enormous mixture of targeting several signaling pathways to have an effects on yield [13]. impact on all aspects of root growth and branching, from Over the years, the effects of genetic factors, plant initiation to post-emergence growth. Nevertheless, it is hormones and nutrients on root growth, root branching interesting that major environmental signals such as

601 [(Figure_1)TD$IG]Opinion Trends in Plant Science Vol.15 No.11

N2

(a) Root symbiotic processes

(Arbuscular) mycorrhizal Nodulation symbiosis

C C + - NH4 PO4

- Pi NO3 + NH4

(b) Root growth, branching and surface Root hairs Lateral root emergence - NO3 - NO3

- PO4

Lateral root initiation NO - Root apical meristem 3 - PO4

- PO4

- PO4

TRENDS in Plant Science

Figure 1. Schematic snapshot of key features of the root system relevant for improved nutrient and water uptake and enhanced growth, and the environmental effect of nutrients, such as nitrate and phosphate. (a) Root symbiotic interactions with other organisms such as fungi and bacteria provide a source of nutrients via uptake and conversion of atmospheric N2 (legumes) (dotted line) or phosphate and nitrate into usable energy. (b) To improve uptake of water and nutrients, plant roots increase in length via a continuously dividing root apical meristem, increase their size through lateral root branches and enlarge their surface with root hairs. While nutrients are taken up to maintain growth through the various highlighted systems, they also directly affect the development of those systems, as indicated for the case of high concentration (broken line).

phosphate and nitrate act on root architecture by modify- corrhizal symbiosis with Glomeromycota fungi species, ing auxin transport and signaling components [39,40]. ubiquitous in soil [41] (Figure 1). The fungal network tremendously increases the root surface area for nutrient Improved uptake and fixation of nutrients uptake; and phosphate, but also nitrogen, are transferred Global phosphate reserves are on their way to total deple- to the plant via active transport mechanisms along the tion and nitrogen is often a limiting factor to plant growth peri-arbuscular membrane, a process that is crucial to [9,10], but a number of root-associated processes that sustain symbiotic interaction [42,43]. Until now, most establish upon mutual chemical signal perception in the agricultural cultivation strategies have searched for the rhizosphere lead to a better fixation of these nutrients. ideal fungal symbiont leading to the highest yield upon Besides the phosphate acquisition through ectomycorrhi- inoculation of the plantlets [44]. However, the signaling zal associations, 80% of land plants obtain important components required to establish and maintain the arbus- mineral nutrition through the ancient arbuscular endomy- cular mycorrhiza symbiosis can be one of the most attrac-

602 Opinion Trends in Plant Science Vol.15 No.11 tive players to manipulate and improve root surface and Box 3. Root responses to symbionts versus pathogens nutrient uptake. The identification of the crucial phos- Growing root systems develop resistance to pathogen attacks by phate transporter provided the first molecular evidence inducing defense response mechanisms, but at the same time they that phosphate uptake in mycorrhized crops could be are able to host micro-organisms, such as (rhizobial) symbionts, manipulated to improve uptake and plant growth [42]. which are not recognized as pathogens. These mechanisms that Related to this, an evolutionarily much younger root maintain symbiosis and do not trigger plant defense, are poorly understood, but surprisingly do not depend on the symbiotic symbiosis exists: root nodulation, which is restricted to performances, i.e. bacterial nitrogenase activity [102]. Besides the legume and actinorhizal plant species, in association with fact that rhizobia exude Nod factor signaling compounds to trigger nitrogen-fixing soil bacteria, Rhizobium or Frankia spp., the host symbiotic response, and arbuscular mycorrhiza fungi a respectively [17,45] (Figure 1). Root nodule symbiosis is comparable Myc factor, there are other extracellular bacterial and favored when legumes or actinorhizal plants are grown in fungal molecules and signals that maintain the symbiotic nature of the interaction. In this respect, it is known that several bacterial the absence of nitrate fertilizer, and therefore encouraged surface polysaccharides play a crucial role in host recognition and to internalize symbiotic bacteria that fix atmospheric ni- infection efficiency [103]. However, most rhizobia also have a Type- trogen in a micro-aerobic environment, such as the inside three secretion system (TTSS) which can positively or negatively of the nodule, providing an unlimited nitrogen source. This regulate symbiosis, by secretion of nodulation outer protein unique capacity allows legumes to propagate well on nu- effectors [104,105]. Proof of concept was shown for the pathogenic Ralstonia strain, for which after acquisition of a symbiotic plasmid, trient-deprived soil, which is beneficial for agricultural use the conversion into a nodulating host for Mimosa was simply to reduce the pollution and environmental damage caused acquired after spontaneous loss of function of a TTSS regulator by fertilization and nitrate loss. Of the total nitrogen added gene [106], thus demonstrating that a pathogenic interaction might to agricultural land, 30–50% is attributable to the legume– easily become beneficial when the key players are known. These rhizobia symbiosis [46]. subtle regulatory differences conversely create opportunities for inefficient, parasitic bacteria that induce root nodules and infect Model legumes, such as M. truncatula and L. japonicus, them, but fix nitrogen only poorly or not at all [80,107]. Apparently were shown to be very useful to study symbiotic signaling such bacterial freeloaders are overlooked by certain crops, making cascades triggered upon perception of compatible organ- them parasitic rhizobia for these actual non-host crops. Research isms in the rhizosphere [17] (see also Boxes 2 and 3). into this area could help us to improve (legume) crops by increasing Because of their protein-rich seeds required for our dietary their susceptibility for beneficial interactions. protein and animal forage, pasture and crop legumes – with soybean, common bean (Phaseolus vulgaris), white L. japonicus Histidine Kinase 1 (LHK1) cytokinin receptor clover (Trifolium repens), alfalfa (Medicago sativa) and pea resulted in spontaneous nodulation, altogether emphasiz- (Pisum sativum) as the most prominent ones – are impor- ing that cytokinin is the hormonal key player in regulation tant crop plants worldwide, second only to grasses in of legume–rhizobium nodulation [49], rather than other economic importance in world agriculture. In addition, hormones such as auxin [50,51]. Moreover, the presence of these plants mostly prevail in nitrate-poor environments active CCaMK kinase, and thereby downstream cytokinin by the presence of compatible bacteria in the root nodules, signaling, was shown to be sufficient for infection and which sustain their life by delivering fixed nitrogen. nodule formation in the root cortex in the absence of Better knowledge and understanding (see also Box 3), as Nod factor molecules and receptors [52]. This new insight well as new insights with potential to improve legume crop also has potential for efficiently transferring the nodula- symbiotic performance and how to transfer symbiosis-re- tion capability to non-nodulating crops, as one could avoid quired signaling cascades to non-nodulating crops, will the required Nod factor-triggered signaling components contribute significantly to sustainable agriculture. In and make use of the already present cytokinin machinery. legumes, the symbiotic pathway is switched on after LysM While by far the most efficient way of bacteria to deliver receptor-like kinase-mediated perception of Nod factors, i.e. nitrogen directly to the roots is through nitrogen fixation in bacterial signaling molecules inducing nodule signaling. the form of root nodule symbiosis, free-living bacteria, such This initial signaling mechanism could be one target to as Azospirillum brasilense, loosely associating with many manipulate and improve, for instance, the host specificity plant species, were also shown to fix nitrogen and increase in legume crops. This is possible via introducing a single plant yield and soil fixation [53]. Furthermore, Acetobacter amino acid variation in the LysM domain, which presum- in the rhizosphere of sugar cane cultivars in Brazil was ably senses or binds the species-specific Nod factor, to allow shown to have specific Nod factor-like penta-lipopolysac- only the best hosts regarding the nitrogen fixation efficiency charide structures that lead to advantageous nitrogen [47] (see also Box 3). This process could also be the target for fixation in these rhizospheres [54]. These successes could implementation into non-nodulating crops, for which Nod be considered to improve plant and yield via factor receptors as such were not defined although most crop encouraging a better associative growth by taking advan- genomes contain large LysM receptor-like kinase families. tage of the environment. However, so far many efforts have failed [12]. However, Nod gene-lacking Bradyrhizobia, which can- Can we avoid genetically modified organisms? not produce or secrete Nod factors, can form nodules on While laboratory-generated modifications of endogenous some Aeschynomene spp. [48]. In this case, bacterially plant genes or of plant-associated organisms are highly produced purine derivates were associated with the induc- valuable, they are not yet accepted worldwide. However, tion of the nodulation process, indicating that a Nod factor- plants adapt to an undesirable environment, thanks to independent pathway can induce nodule formation. In their enormous phenotypic plasticity, in contrast to ani- addition, a recent gain-of-function mutation study in the mals, which just move away from an undesirable environ-

603 Opinion Trends in Plant Science Vol.15 No.11 ment. This means that within one plant species, ecotypes their root branching potential [71]; and also for strigolac- can display enormous variability in their root systems, and tones, the novel class of shoot branching hormones [72], within one ecotype the root architecture can vary according whose role in root branching has not yet been investigated. to environmental conditions [6,55,56]. Hence, one can In addition, a number of species display specialized roots, make use of genetic traits and diversity that are present collectively called ‘cluster roots’, to maximize phosphate in natural populations, as is the case for wild barley acquisition from low fertility soils, such as proteoid roots or (Hordeum vulgare subsp. spontaneum K. Koch) [57],by dauciform roots [9,73]. Further research might be required analyzing various ecotypes and identifying the responsible to develop some of these into new model species. quantitative trait loci (QTLs), which can subsequently be Communication between root and shoot and vice versa combined through classical breeding. Yet, in this respect, to balance the carbon supply with nutrient acquisition, to only very few QTLs for root architecture have been cloned modulate shoot and root architecture, and to optimize to date [7,58,59]. Because drastic loss-of-function muta- plant fitness requires further investigation. Some hormon- tions associated with large effect QTLs are rarely relevant al and molecular aspects are known, such as the impor- in nature, one way forward is the isolation of differentially tance of shoot-derived auxin for lateral root emergence active, functional alleles, as was recently exemplified by [74], the autoregulation of nodulation via the shoot- the identification of a hyperactive allelic variant of BRE- expressed receptor-like kinase HYPERNODULATION VIS RADIX (BRX) [60]. In addition, we should not only ABERRANT ROOT FORMATION 1 (HAR1) and peptides select for plants with the most advanced root architecture or other molecules that signal between root and leaves [75], phenotype, but also for those with the best ability to adapt and the root as a source of signals to control shoot archi- root development to the appropriate growth conditions. In tecture, such as strigolactones [72] and a BYPASS1 contrast to QTL analysis on plant–pathogen interactions, (BPS1)-dependent signal [76,77]. When investigated in the identification of symbiotic traits in legumes seems to be more detail, these pathways will provide new insights in less agriculturally attractive and therefore still largely how plant parts communicate to balance growth and de- unexplored [61]. Nevertheless, the QTL mapping for sym- velopment. biotic traits will be more accessible as legume genome Lateral roots and nodules are secondary organs altering sequences are completed [62,63]. The first indications for the structure and hormonal landscape of the root. It has a positive relation in genetic trait variability between root always been assumed that the nodule developmental pro- development, nodule establishment and nitrogen nutrition gram had been hijacking (lateral) root formation molecular was recently demonstrated in pea and hints towards po- components. For example, the LATERAL ROOT ORGAN- tential combinatorial QTLs to improve the root system DEFECTIVE (LATD)/NUMEROUS INFECTIONS AND size, nodule number and nutrition in the same plant [64]. POLYPHENOLICS (NIP) gene encodes a transporter of In future, various improvements will facilitate the iden- the NRT1 family, and mutants are defective in several tification and cloning of QTLs associated with complex stages of (lateral) root and nodule development, demon- traits [65]. However, it should be noted that QTLs in strating the link between both developmental pathways one background might not be successful in another [66]. [78]. Further insights into these signalling mechanisms would allow connecting these processes to root architecture Future research directions and to create models how to optimize root architecture for One downside of changing certain traits is that it often growth and nutrient uptake. negatively affects other traits, and at the moment it is Despite the importance of detailed ‘single gene’ analy- unclear how to predict this trade-off. For example improving ses, which are of the outmost importance for the transla- the root system to deal with one condition might lead to a tion of knowledge into crop species, ‘omics’ analyses and negative outcome under other conditions [67],orimproving systems biology approaches will remain important to un- the yield under drought can lead to a decrease in root derstand whole processes and molecular networks. For biomass [66,68]. To solve this, we will need to combine example, recent network analysis resulted in the identifi- desirable traits and avoid undesired qualities, and make cation of LATERAL ROOT STIMULATOR 1 (LRS1), which use of new modeling technologies to dissect complex pheno- affects lateral root emergence and/or elongation [79]. types and to predict which combinations are favorable [69]. Furthermore, besides the well-studied and rather con- This requires high-quality phenotype data, and the complex- served mechanisms for early recognition, root nodule sym- ity of a root system needs to be captured in detail, e.g. biotic functionality mainly depends upon bacterial including numbers of initiated, arrested and outgrown lat- endocytosis during infection, intracellular residence and eral and adventitious roots; branching pattern; higher order fixation capacity to result in a compatible interaction (see branching; total root length and root biomass. Recent tech- also Box 3) [80]. Therefore, transfer of beneficial mechan- nologies now allow the generation of such data for Arabi- isms to non-nodulating crops implies a better molecular dopsis and crop root systems [7,55,70]. In addition to lateral understanding of the signals required for functional root roots, root hairs develop from root epidermal cells to aid nodule symbiosis at later stages. The research addressing nutrient and water uptake, and to interact with microbial these questions is still in its infancy, but points to diver- symbionts [17,25–27] (Figure 1). These and other aspects, gent signaling mechanisms among different legume spe- such as conductivity and root growth rates, should also be cies, complicating our search for the right genes [80]. In the considered for modeling and improving the root system. end, the implementation of signaling components into non- New potential for research could be found in the various legume crops will not only demand profound in-depth shoot branching genes that have not been analyzed for research on the targeted crops itself, but also implementa-

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