Using membrane transporters to improve crops for sustainable food production

Abstract

With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land.

Of The Present Global Population Of Seven Billion People, Almost One billion are undernourished and lack sufficient protein, fats and carbohydrates in their diets¹. An additional billion people are malnourished because their diets lack required micronutrients such as iron, zinc and vitamin A (ref. 2). These dietary deficiencies have an enormous negative impact on global health, resulting in increased susceptibility to infection and diseases, as well as increasing the risk of significant mental impairment³. During the next four decades, an expected additional two billion humans will require nutritious food. Along with growing urbanization, increased demand for protein in developing countries, coupled with impending climate change and population growth, will impose further pressures on agricultural production⁴. Global demand for food is predicted to increase by 40% by 2030 (ref. 4). Innovative solutions are required to increase production on the land currently used for agriculture, because we are already close to the sustainable limit of 15% of the Earth’s surface that can be exploited for crop production⁵.

Analysis of crop yields globally shows that in those developing regions where humans are most susceptible to malnutrition, the availabilities of inorganic nutrients and water are the principal factors that determine crop productivity^6,7. Simply increasing inorganic fertilizer use and water supply or applying organic farming systems to agriculture⁸ will be unable to satisfy the joint requirements of increased yield and environmental sustainability. Increasing food production on limited land resources will rely on innovative agronomic practices coupled to the genetic improvement of crops⁹.

Transport proteins embedded within membranes are key targets for improving the efficiency with which plants take up and use water and nutrients. These proteins not only transport mineral nutrients and control drought tolerance but are also essential for moving sucrose, the energy currency of plants, to where it is needed. Furthermore, transporters are also central to mechanisms that allow plants to tolerate adverse environments such as saline or acid soils. Advances driven by physiology, genetics and biophysics over the past 20 years have dramatically improved our understanding of the molecular basis of plant nutrition and how plants respond to stress. Genome sequencing and the development of experimental systems for studying transporter function have allowed many of the major families of membrane transporters to be characterized. Next-generation sequencing is leading to an understanding of how the natural genetic diversity of plant membrane transporters can be exploited for agriculture, whether by marker-assisted breeding or through genetic engineering. Breakthrough approaches involving transcriptional-activator-like effectors and genome editing¹⁰ can provide non-genetically modified (non-GM) yet molecularly directed rapid solutions to crop improvement.

Here we report on findings demonstrating that understanding the biology of plant membrane transporters can be a key contributor to the goal of global food security. We discuss examples where fundamental research is already being translated into practical applications such as enhancing the micronutrient content of grain and improving the plant tolerance to saline and acidic soils. We further discuss potential applications linked to breakthroughs in basic research that are yet to be applied to crop plants. This Perspective reviews the extent to which the rapid advances in plant transport research address global aspects of food security, and how we can potentially reduce the time between trait identification in the laboratory and exploitation in the field.

Transporters, stress resistance and yield

Aluminium-tolerant crops for acid soils

Acid soils comprise 30% of Earth’s ice-free land and thus constrain agricultural production, given that only a small proportion of these soils is suitable for crops¹¹. At soil pH values above 5, aluminium exists in the soil in non-toxic complexed forms. However, when soils are acidic, Al³⁺ ions are freed in the soil, resulting in plant toxicity. Once in the soil solution, Al³⁺ damages the root tips of susceptible plants and inhibits root growth, which impairs the uptake of water and nutrients. Natural genetic variation in Al³⁺ tolerance exists within major cereal crops. The efflux of organic anions from roots was discovered to be a naturally occurring tolerance mechanism of several species¹². Transport proteins are central to this mechanism, with members of two families of transport proteins responsible for exporting the organic anions from inside root cells to the external medium surrounding roots. The organic anions secreted by roots chelate Al³⁺ into a non-toxic form, thus protecting the sensitive tips and allowing the roots to grow unimpeded (Fig. 1).

Figure 1. Engineering plants for enhanced aluminium (Al³⁺) tolerance.

The photograph shows barley seedlings grown on an acid soil that contains high concentrations of toxic Al³⁺. One seedling has been genetically engineered with an Al³⁺-tolerance transporter gene from wheat (TaALMT1), whereas the other seedling is the non-transgenic parental line (wild type, WT). The diagram shows the TaALMT1 anion channel (blue structure) embedded within the plasma membrane of apical root cells. In acid soils, Al³⁺ activates TaALMT1 (dashed line) resulting in malate efflux into the apoplast (cell wall) external to the cytoplasm. Malate molecules (OA⁻, yellow circles) bind Al³⁺ in the apoplast to protect cells from aluminium toxicity at the root apex. The diagram is modified from figure 2 in ref. 92.

In wheat, the Triticum aestivum aluminium-activated malate transporter 1 gene TaALMT1 encodes an Al³⁺-gated anion channel that facilitates malate efflux from roots¹³ (Fig. 1). Molecular markers based on the TaALMT1 gene can be used in marker-assisted breeding to select for Al³⁺ tolerance in wheat germplasm. The TaALMT1 gene itself can be used to genetically modify susceptible species for improved Al³⁺ tolerance. When expressed in barley, one of the most Al³⁺-sensitive cereal crops, TaALMT1 confers substantially improved grain yields in acid soil¹⁴ (Fig. 1). Similarly, a unique subgroup of the large family of plant multidrug and toxic compound extrusion (MATE) transporters mediates citrate efflux from root cells^15,16. In sorghum¹⁶, barley¹⁷, and maize¹⁸, MATE transporters located at the root tip confer Al³⁺-activated citrate efflux and represent the primary Al³⁺ tolerance proteins. Genetic studies in sorghum recently identified markers associated with Al³⁺-tolerant alleles of the Sorghum bicolor MATE gene SbMATE. These markers have been used by breeders to introgress rapidly the most favourable SbMATE alleles into sorghum germplasm, which is currently being field-tested in acid soils¹⁶.

Although genes encoding organic anion transporters are already established as a means of enhancing the Al³⁺ tolerance of crops, other recently discovered mechanisms provide additional options. For instance, rice is the most Al³⁺ tolerant of the cereal crops and uses mechanisms distinct from the efflux of organic anions. One of these mechanisms involves transporters working in concert ultimately to sequester Al³⁺ into the vacuole, thus removing Al³⁺ from mainstream metabolism^19,20. Genetic analysis has identified susceptible and tolerant variants of one of the transporter genes (natural resistance-associated macrophage protein aluminium transporter 1, Nrat1) that explains a large proportion of the variation in Al³⁺ tolerance within the rice aus subpopulation. This finding provides a promising tool for marker-assisted breeding²¹.

The discovery of transporters that mediate Al³⁺ tolerance has identified two principal strategies that plants use to deal with this toxic cation. In one strategy, Al³⁺ is excluded from cells by chelating the toxic ion external to plants and in the other Al³⁺ is sequestered within cells in the vacuole. The genes encoding these transporters can be used to develop Al³⁺-tolerant crops and represent an important component—along with management practices such as soil liming to increase soil pH—of a strategy for improving yields on acid soils.

HKT transporters improve salt tolerance

Approximately 7% of the world’s land including agricultural lands is affected by either salinity or sodium toxicity. Production in over 30% of irrigated crops and 7% of dryland agriculture worldwide is limited by salinity stress. Crop irrigation is increasing soil salinity, owing to trace amounts of salt in irrigation waters. Plant plasma membrane transporters in the HKT family transport sodium (Na⁺) and potassium (K⁺) (ref. 22) and play an essential part in salt tolerance²³. Research in the reference plant Arabidopsis showed that the ‘class 1’ HKT transporters are Na⁺ selective and protect plant leaves from salinity stress by prohibiting toxic sodium over-accumulation in leaves²³. Class 1 HKT transporters are expressed in veins²³ that connect nutrient flux between roots and leaves. These transporters are expressed in the living cells surrounding the xylem, which are vessels that carry nutrients and water to the leaves. Class 1 HKT transporters remove excess Na⁺ from the xylem in Arabidopsis and rice, thereby keeping Na⁺ below toxic levels in the photosynthetic leaf tissues^24–26 (Fig. 2). Analogous mechanisms have been demonstrated in wheat for the HKT1;4 and HKT1;5 genes^27,28 (Fig. 2). Remarkably, the recent introgression of an ancestral form of the HKT1;5 gene from the more Na⁺-tolerant wheat relative Triticum monococcum into susceptible commercial durum wheat (Triticum turgidum ssp durum) increased grain yields on saline soil by 25% in the field, illustrating the immense potential of this mechanism²⁸.

Figure 2. HKT transporter-mediated salt tolerance in plants.

The drawing illustrates the function of class I HKT transporters in protecting plants from salinity stress. These HKT transporters mediate Na⁺ unloading from the xylem under salinity stress, which prevents Na⁺ over-accumulation in leaves, thereby protecting photosynthetic organs. An example of this mechanism in wheat plants is shown.

Some crops are salt tolerant through the effective sequestration of Na⁺ in leaf vacuoles by Na⁺/H⁺ antiporters²⁹. Specific ‘class 2’ HKT transporters³⁰ mediate cation influx into roots³¹. These class 2 HKT transporters, together with transporters that sequester sodium and potassium in the vacuole^32,33, have the potential to improve the production of cereals such as barley, a species that copes with high Na⁺ loads in leaves by compartmentation in the vacuole³⁴. Thus combining (pyramiding) HKT transporter traits with vacuolar Na⁺ sequestration mechanisms provides a potentially powerful platform for molecular breeding and transgenic approaches to improve the salinity tolerance of crops.

SWEET transporters and pathogen resistance

Photosynthesis in leaves produces sugars, which are distributed through the veins to support growth of roots, meristems and seeds. The translocation rates and relative distribution critically determine the yield potential of crops. Sucrose is a key energy-carrying molecule that also acts as the driver for translocation of all other nutrients and signalling molecules in veins, but until recently, the molecular players were unknown. In the early 1990s the sucrose-proton co-transporter SUT1 was identified as the key transporter that loads sugar into veins³⁵, yet the mechanism for sucrose release from the leaf cells that synthesize it remained elusive. Recently, the ‘SWEET’ sugar transporters were identified with the help of sugar sensors based on Förster (fluorescence) resonance energy transfer technology^36,37. Known crop genomes possess about 20 SWEET genes. SWEETs are plasma membrane proteins located in the phloem parenchyma, a cell type inside the veins that exports sucrose to the SUT1 sugar loaders (Fig. 3). The import and export of sucrose from vein cells is controlled by hormones, turgor feedback and sugar levels³⁸. Knowledge of this machinery could provide a new starting point to engineer yield by modifying energy and carbon distribution within the plant.

Figure 3. The role of SWEET sugar transporters in efflux of sucrose into the cell-wall space and induction by pathogenic bacteria.

a, SWEETs (red) localized in the phloem parenchyma (a cell type of the plant vasculature), export sucrose produced by photosynthesis into the cell wall, from where it is loaded actively, with the help of the transporter SUT1 and energized by H⁺-ATPases into the actual conduits, the sieve element companion cell complex for translocation to seeds. Photosynthesis mainly occurs in the palisade parenchyma. b, The role of SWEETs as the ‘Achilles’ heel’ (susceptibility factors) of host plants during pathogen infection. SWEETs are induced directly as a consequence of the injection of transcriptional-activator-like effectors from pathogens via type III secretion systems into the infected plant cell, leading to release of sugars as a critical source of nutrition for the pathogens.

Notably, SWEET sugar efflux transporters have been identified as pathogen resistance loci, leading to a new understanding of disease development in plants^39–42. The growth of pathogens in leaves and stems depends on nutrient supply from their plant hosts. Blight bacteria directly induce SWEET gene expression in rice in infected cells through transcriptional-activator-like effectors (bacterial transcription factors that directly target SWEET promoters). Inhibiting the induction of SWEET genes with an innovative technology such as chromosomal editing of the promoters of SWEETs with TALENs (artificial transcriptional-activator-like effector nucleases) or through cell-specific expression of microRNAs in cells outside the phloem has now enabled blight resistance to be engineered in rice^43,44. The discovery of these key players in combination with TALENs promises new ways of engineering both crop yield and pathogen resistance, without the introduction of foreign genetic material, to produce plants with significantly improved performance in the field.

Human health and plant nutrition

Pumping iron and zinc

Over two billion people suffer from iron and zinc deficiencies because their plant-based diets are not a sufficiently rich source of these essential elements. Developing crop cultivars with increased micronutrient concentrations, an approach known as biofortification, is challenging because metal ion concentrations in various tissues and compartments are maintained within narrow physiological limits by coordinated uptake, translocation and storage. Furthermore, for crops like rice, removal of the outer layers of the grain during polishing essentially removes all of the micronutrients, leaving only the starchy endosperm. By expressing key genes involved in the mobilization of micronutrients from the soil to the seed, scientists have biofortified rice, a staple food consumed by half the world population every day (Fig. 4). Enhancing iron translocation through overproduction of the metal chelator nicotianamine and phytosiderophores^45–47 or enhancing iron influx into the endosperm by means of the iron-nicotianamine transporter Oryza sativa yellow-stripe-like-2 (OsYSL2)⁴⁸, has resulted in greenhouse-grown rice with three- to fourfold higher levels of iron (Fe) in polished grain. Combining overproduction of nicotianamine with enhanced expression of the iron storage protein ferritin increased the iron content more than sixfold⁴⁹, and combining all three approaches has resulted in paddy-field-grown polished rice with Fe concentrations 4.4-fold higher than those found in non-transgenic seeds, with no yield penalty⁵⁰. Although these results are impressive and bring iron levels close to those recommended by nutritionists, only a handful of studies have tested whether these enhanced levels of nutrients are bioavailable. Most encouragingly, enhancing the nicotianamine concentration does increase the levels of bioavailable iron⁴⁶ and zinc⁵¹ in polished rice.

Figure 4. Iron transport in rice.

Rice takes up iron from the soil as Fe³⁺ deoxymugineic acid (DMA) by the OsYSL15 transporter. Rice also uses the OsIRT1 transporter to take up Fe²⁺, which is abundant in submerged and anaerobic conditions. DMA, which is the primary phytosiderophore that aids in iron transport, is synthesized from S-adenosyl methionine through three sequential enzymatic reactions mediated by nicotianamine synthase (NAS), nicotianamine aminotransferase (NAAT), and DMA synthase (DMAS), and then secreted by the efflux transporter TOM1 to solubilize iron in the soil. Nicotianamine, which is the biosynthetic precursor of DMA, is a chelator of divalent metals and plays a part in translocation of metals within plants. Nicotianamine is secreted into the cell wall by the nicotianamine efflux transporter ENA1. The iron–nicotianamine transporter OsYSL2 mediates iron influx into rice grains. The photograph shows iron staining (blue coloration) of a rice seed (inset shows rice seeds). Iron is mainly localized to the embryo and the outer layers of the grain.

Vacuolar sequestration is another mechanism to enhance the concentrations of iron and zinc (Zn) in seeds⁵². Transporters belonging to several different families transport metals between the cytoplasm and the vacuole^53–55, including the Arabidopsis vacuolar iron transporter VIT1 protein, which is highly expressed in developing seeds and transports iron and manganese into the vacuole⁵⁴. Disruption of the rice VIT orthologues (OsVIT1 and OsVIT2) increases Fe/Zn accumulation in rice seeds and decreases Fe/Zn in the source organ flag leaves, probably because VIT genes are highly expressed in rice flag leaves. Without a sink, there is enhanced Fe/Zn translocation to the seed, providing another strategy with which to biofortify Fe/Zn in staple foods⁵⁶. Metal tolerance protein 1 (MTP1) also transports divalent cations into the vacuole and is another promising candidate for use in biofortification⁵⁷. Thus several strategies are being used to enhance iron and zinc micronutrients in edible plant tissues, but more improvements are needed. We can use our growing knowledge of the transporters that take up micronutrients from the soil, such as iron-regulated transporter 1 (IRT1)⁵⁸, the major entry point for Fe in many plant species. Enhanced nutrient content is a crucial goal in the light of the world’s growing population and the central roles of staple crops in human diets.

Enhancing phosphate use efficiency

Phosphorus (P) is a macro-element that is essential for plant growth and of vital importance to crop yield. The availability of inorganic P, or orthophosphate (the only form of P directly accessible to plants), is influenced by the biogeochemical properties of the soil and limits crop production on nearly 70% of the world’s agricultural soils⁵⁹. Consequently, global crop production depends on orthophosphate fertilizers, which are produced from rock phosphate, a finite, non-renewable mineral resource (Fig. 5a). Only 20–30% of the P fertilizer applied is used by cultivated plants and at the current rate of use, it is estimated that rock phosphate reserves will be consumed within the next 70–200 years⁶⁰, so ensuring the sustainable use of orthophosphate is of paramount importance for human nutrition. Improving orthophosphate acquisition and use-efficiency in plants is a complex problem and recent solutions have included modifications to root growth and architecture^61,62, and novel engineering strategies to use alternative P sources⁶⁰. An understanding of plant transporter proteins may offer additional approaches. Plants possess several families of orthophosphate transporter proteins, and both high- and low-affinity transporters are important for orthophosphate uptake into roots^63,64. Phosphate transporters are also critical for orthophosphate distribution throughout the plant, and for remobilization between source and sink tissues^65,66. A phosphate efflux transporter (PHO1), essential for orthophosphate transfer to the shoot, is a major player in the regulation of orthophosphate homeostasis⁶⁷ and may provide strategies for optimizing orthophosphate distribution within plants.

Figure 5. Global phosphate availability and nitrate sensing.

a, Global distribution map of reserves of rock phosphate. The percentage of effective reserves is illustrated (data from ref. 93). b, Dual functions of the nitrate transporter and nitrate sensor, CHL1, in nitrate uptake and sensing. Photographs show Arabidopsis seedlings at low (left) and high (right) nitrate. At low nitrate, CHL1 is phosphorylated at a specific amino acid, T101, converting it to a high-affinity nitrate uptake transporter, enabling nitrate accumulation at limiting soil nitrate concentrations. At high nitrate, the T101 amino acid is not phosphorylated, and CHL1 functions as a low-affinity transporter.

In addition to the direct acquisition of orthophosphate from soil, most crop species have the capacity to form symbiotic associations (called arbuscular mycorrhizae) with soil fungi. These fungi capture orthophosphate through extensive hyphal networks and deliver it to symbiotic compartments in the root, where plant orthophosphate transporters transfer this ion into the root cells. Plant symbiotic orthophosphate transporter function is essential for this process and is also required to maintain the symbiosis^68,69. Phosphate transporters are important targets for breeding plants with improved orthophosphate acquisition and use-efficiency, and that benefit maximally from their fungal symbionts.

Nitrate sensing and transport

The application of nitrogen (N) fertilizers has greatly increased crop yields and alleviated hunger over the past five decades. However, N fertilizer production consumes 1% of global energy usage and poses the highest input cost for many crops. Nevertheless, only 30% to 50% of the N fertilizer applied is used by plants. The remainder can lead to production of the greenhouse gas N₂O, or to eutrophication of aquatic ecosystems through water run-off. Therefore, enhancing crop nitrogen utilization efficiency is an important goal⁷⁰. For most crops, nitrate is the primary nitrogen source and so enhancing nitrate uptake is one strategy for improving nitrogen utilization efficiency.

Multiple nitrate uptake transporters of the NRT1 and NRT2 families work together to enable nitrogen uptake in plants^71,72. Most NRT1 transporters are low-affinity nitrate transporters, meaning that they function mainly when nitrate is abundant. In contrast, chlorate resistant 1 (CHL1, also known as NRT1.1) is a dual-affinity nitrate transporter involved in nitrate acquisition^73,74. A recent study found that CHL1 also functions as a nitrate sensor, thus regulating nitrate-induced gene expression⁷⁵, which implies that plants use this transporter to monitor changes in external nitrate concentration to trigger proper metabolic acclimation. CHL1 has therefore become a paradigm for how nutrient transporters may also serve as nutrient sensors, and how optimization of transport and signalling can be used simultaneously to improve nutrient efficiency.

Using dual-affinity binding, and with the help of two protein kinases (CIPK23 and CIPK8), CHL1 senses a wide range of nitrate concentration changes in the soil to alter its own transport properties^75,76 (Fig. 5b). CHL1 and NRT2.1 are also important for nitrate-regulated root development^77,78. Vigorous root development is important for plants to compete for nutrients and sustain crop yield^61,79. Therefore, nitrate transporters and other proteins that regulate nitrate uptake and sensing provide potential tools for engineering crops with tailored N uptake activity, N metabolism and improved root growth for enhanced nitrogen-use efficiency and reduced-N-fertilizer requirements.

Future outlook

Our knowledge of the molecular nature and regulation of transporters has expanded vastly over the past twenty years. As shown in the examples here, fundamental research into transport mechanisms in plants is leading to rapid innovations for improving yields, extending the range of arable land that can be used for crops and improving the performance of plants under stress. This research also points to new solutions for more sustainable use of limited soil nutrients and for enhanced human nutrition through biofortification.

Recent advances on other plant transporters add to the list of potentially innovative applications in agriculture. For example, plants in the Brassica family, which includes oilseed rape (canola) and mustard, produce glucosinolates, which are potent defence compounds against herbivores and plant pathogens. A transporter controls the distribution of glucosinolates in Arabidopsis⁸⁰ and may be engineered to enhance herbivore resistance, as a way of reducing the application of pesticides. In another example, toxic heavy metal and arsenic accumulation in edible plant tissues, including rice in the United States, poses a threat to human health⁸¹. Plant transporters have recently been identified that control toxic metal and arsenic accumulation in seeds and other tissues^82–84, pointing directly to potential applications for developing plants with reduced levels of toxicant accumulation in grain and other edible tissues. Rice with nearly cadmium-free grain has been produced by identifying cadmium transporters and also using a molecular marker to select for genotypes that accumulate low cadmium concentrations⁸⁵.

Drought tolerance and desiccation avoidance by plants is critical for conserving water during drought periods. Plants lose over 90% of their water by transpiration from stomatal pores in the epidermis of leaves. Stomatal pores are also the gateways for CO₂ intake into plants for photosynthesis and have a key role in determining the water-use efficiency of plants. Research has shown that modification in the expression level of ion channels in guard cells (which control the opening and closing of stomatal pores) can be used to reduce water loss, enhance water-use efficiency and regulate efficient CO₂ intake for photosynthesis^86–88. Moreover, transporters for the plant abiotic stress resistance hormone, abscisic acid, have been identified^89,90, and these may be used to target drought resistance responses. Targeting drought tolerance to particularly drought-sensitive tissues or organs during particularly susceptible stages of reproductive development (for example, grain filling and pollen meiosis) could become an important strategy during prolonged droughts and other predicted consequences of climate change.

Transport processes are key to photosynthesis. Since Peter Mitchell’s groundbreaking ‘chemiosmotic hypothesis’ in 1961, the relevance of transporters in photosynthesis has become abundantly clear. Major advances have been made in identifying metabolite transporters across chloroplast membranes⁹¹. Yet many of the key transporters in chloroplast membranes remain unidentified. Discovery of the many predicted transporters in subcellular compartments, specifically in chloroplasts, has potential for improving energy capture.

A major challenge in future agriculture is establishing which genetic traits can be combined, or ‘pyramided’, without adversely affecting yield. Many transport processes reviewed here enhance plant performance via defined functions in specific tissues or cell types, so these may be particularly amenable to pyramiding. For instance, salinity tolerance that operates by removal of toxic sodium ions from the xylem sap^23–25,28 could be combined with traits that enhance sequestration of sodium into vacuoles^32,33, to confer additional salt tolerance. More work will be needed to determine whether or not traits will be compatible when combined. Moreover, many fundamental mechanisms for essential transport processes remain to be uncovered and many essential transporters undoubtedly remain to be discovered. Therefore, knowledge-targeted pyramiding of traits will require future advances in fundamental research into plant membrane transport processes.

Recent advances have identified plant membrane transporters and underlying mechanisms that increase the stress resistance and yield of staple crops. We expect that research into fundamental mechanisms of plant membrane transport processes will continue to produce surprises and breakthroughs that will provide new avenues towards a more sustainable and productive agriculture in the face of impending challenges.