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Climate change is challenging food production worldwide. We propose a strategy to accelerate the improvement of a nutritious resilient crop to meet the future food production demands in a changing climate., Crops tolerant to drought and salt stress may be developed by two approaches. First, major crops may be improved by introducing genes from tolerant plants. For example, many major crops have wild relatives that are more tolerant to drought and high salinity than the cultivated crops, and, once deciphered, the underlying resilience mechanisms could be genetically manipulated to produce crops with improved tolerance. Secondly, some minor (orphan) crops cultivated in marginal areas are already drought and salt tolerant. Improving the agronomic performance of these crops may be an effective way to increase crop and food diversity, and an alternative to engineering tolerance in major crops. Quinoa (Chenopodium quinoa Willd.), a nutritious minor crop that tolerates drought and salinity better than most other crops, is an ideal candidate for both of these approaches. Although quinoa has yet to reach its potential as a fully domesticated crop, breeding efforts to improve the plant have been limited. Molecular and genetic techniques combined with traditional breeding are likely to change this picture. Here we analyse protein-coding sequences in the quinoa genome that are orthologous to domestication genes in established crops. Mutating only a limited number of such genes by targeted mutagenesis appears to be a promising route for accelerating the improvement of quinoa and generating a nutritious high-yielding crop that can meet the future demand for food production in a changing climate.

Based on recent in vitro data, a relatively large number of the plant Nitrate transporter 1/Peptide transporter Family (NPF) proteins has been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities towards organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis identified distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.

The latest major group of plants to evolve were the grasses. These became important in the mid-Paleogene about 40 million years ago. During evolution, leaf CO2 uptake and transpirational water loss were optimized by the acquisition of grass-specific stomatal complexes. In contrast to the kidney-shaped guard cells (GCs) typical of the dicots such as Arabidopsis, in the grasses and agronomically important cereals, the GCs are dumbbell shaped and are associated with morphologically distinct subsidiary cells (SCs). We studied the molecular basis of GC action in the major cereal crop barley. Upon feeding ABA to xylem sap of an intact barley leaf, stomata closed in a nitrate-dependent manner. This process was initiated by activation of GC SLAC-type anion channel currents. HvSLAC1 expressed in Xenopus oocytes gave rise to S-type anion currents that increased several-fold upon stimulation with >3 mM nitrate. We identified a tandem amino acid residue motif that within the SLAC1 channels differs fundamentally between monocots and dicots. When the motif of nitrate-insensitive dicot Arabidopsis SLAC1 was replaced by the monocot signature, AtSLAC1 converted into a grass-type like nitrate-sensitive channel. Our work reveals a fundamental difference between monocot and dicot GCs and prompts questions into the selective pressures during evolution that resulted in fundamental changes in the regulation of SLAC1 function. Schäfer et al. report that guard cells of the cereal crop barley require nitrate for ABA-induced stomatal closure—a feature accomplished by the guard cell anion channel HvSLAC1. Nitrate-dependent gating of HvSLAC1 and other monocot SLAC1-type anion channels evolved from a TMD3 tandem motif after the split between monocots and dicots.

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge. DOI: http://dx.doi.org/10.7554/eLife.19466.001, eLife digest All living cells are surrounded by membranes that protect them from the external environment. The membrane contains proteins called transporters, which move nutrients and other molecules (known as substrates) across the membrane. A variety of transporters have evolved to move the hundreds of thousands of different substrates found in nature. Plant cells make many different compounds to protect themselves from pests and diseases. A group of transporters known as the NPF family move some of these compounds across the cells outer membrane. The types of substrates they transport vary in different plants. In cassava, for example, NPF transporters move compounds called cyanogenic glucosides, which are poisonous to humans and other animals. On the other hand, NPF transporters in another plant called Arabidopsis thaliana can move bitter-tasting compounds called glucosinolates. The process that makes glucosinolates in plants evolved from the process that makes cyanogenic glucosides. Can transporters evolve the ability to move a new substrate before or after that substrate first appears? To answer this question, Jørgensen et al. studied the NPF family in A. thaliana, cassava and another plant called papaya that makes both cyanogenic glucosides and glucosinolates. The experiments suggest that NPF transporters able to move both cyanogenic glucosides and glucosinolates evolved before plants evolved the ability to make glucosinolates. Later in evolution, these multi-specific transporters specialized to only move glucosinolates. Jørgensen et al. also show that early glucosinolate transporters could move a broad variety of glucosinolates but later evolved to only transport particular types. These findings show how transporters and the processes that make compounds in cells may evolve together. A future challenge will be to understand the molecular changes in a transporter that make it specific for a certain substrate. This may help researchers to develop new ways of controlling the amount of toxic compounds in crops we eat by manipulating how the compounds are transported. DOI: http://dx.doi.org/10.7554/eLife.19466.002

This protocol describes how to order and directly assemble uracil-containing non-clonal DNAfragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal ofmaking synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol ishighly efficient and would be compatible with uracil-containing non-clonal DNA fragments obtained fromany synthesizing company. The protocol drastically reduces time and handling between receiving thesynthesized DNA fragments and transforming with vector and DNA fragment(s).

Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties.In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides ( Jørgensen et al., 2017 ), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.

The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.

Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

Little is known about how plants regulate transporters of defense compounds. In A. thaliana, glucosinolates are transported between tissues by NPF2.10 (AtGTR1) and NPF2.11 (AtGTR2). Mining of the PhosPhat4.0 database showed two cytosol exposed phosphorylation sites for AtGTR1 and one membrane-buried phosphorylation site for AtGTR2. In this study, we investigate whether mutation of the two potential regulatory sites of AtGTR1 affected transport of glucosinolates in Xenopus oocytes. Characterization of AtGTR1 phosphorylation mutants showed that phosphorylation of AtGTR1 - at the two reported phosphorylation sites - is not directly involved in regulating AtGTR1 transport activity. We hypothesize a role for AtGTR1-phosphorylation in regulating protein-protein interactions.