Arbuscular-Mycorrhizal Symbiosis in Medicago Regulated by the Transcription Factor MtbHLHm1;1 and the Ammonium Facilitator Protein MtAMF1;3.

Root systems of most land plants are colonised by arbuscular mycorrhiza fungi. The symbiosis supports nutrient acquisition strategies predominantly associated with plant access to inorganic phosphate. The nutrient acquisition is enhanced through an extensive network of external fungal hyphae that extends out into the soil, together with the development of fungal structures forming specialised interfaces with root cortical cells. Orthologs of the bHLHm1;1 transcription factor, previously described in soybean nodules (GmbHLHm1) and linked to the ammonium facilitator protein GmAMF1;3, have been identified in Medicago (Medicago truncatula) roots colonised by AM fungi. Expression studies indicate that transcripts of both genes are also present in arbuscular containing root cortical cells and that the MtbHLHm1;1 shows affinity to the promoter of MtAMF1;3. Both genes are induced by AM colonisation. Loss of Mtbhlhm1;1 expression disrupts AM arbuscule abundance and the expression of the ammonium transporter MtAMF1;3. Disruption of Mtamf1;3 expression reduces both AM colonisation and arbuscule development. The respective activities of MtbHLHm1;1 and MtAMF1;3 highlight the conservation of putative ammonium regulators supporting both the rhizobial and AM fungal symbiosis in legumes.

Genome Editing for Sustainable Agriculture in Africa.

Sustainable intensification of agriculture in Africa is essential for accomplishing food and nutritional security and addressing the rising concerns of climate change. There is an urgent need to close the yield gap in staple crops and enhance food production to feed the growing population. In order to meet the increasing demand for food, more efficient approaches to produce food are needed. All the tools available in the toolbox, including modern biotechnology and traditional, need to be applied for crop improvement. The full potential of new breeding tools such as genome editing needs to be exploited in addition to conventional technologies. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing has rapidly become the most prevalent genetic engineering approach for developing improved crop varieties because of its simplicity, efficiency, specificity, and easy to use. Genome editing improves crop variety by modifying its endogenous genome free of any foreign gene. Hence, genome-edited crops with no foreign gene integration are not regulated as genetically modified organisms (GMOs) in several countries. Researchers are using CRISPR/Cas-based genome editing for improving African staple crops for biotic and abiotic stress resistance and improved nutritional quality. Many products, such as disease-resistant banana, maize resistant to lethal necrosis, and sorghum resistant to the parasitic plant Striga and enhanced quality, are under development for African farmers. There is a need for creating an enabling environment in Africa with science-based regulatory guidelines for the release and adoption of the products developed using CRISPR/Cas9-mediated genome editing. Some progress has been made in this regard. Nigeria and Kenya have recently published the national biosafety guidelines for the regulation of gene editing. This article summarizes recent advances in developments of tools, potential applications of genome editing for improving staple crops, and regulatory policies in Africa.

Genome-Wide Association Study of Phytic Acid in Wheat Grain Unravels Markers for Improving Biofortification.

Biofortification of cereal grains offers a lasting solution to combat micronutrient deficiency in developing countries where it poses developmental risks to children. Breeding efforts thus far have been directed toward increasing the grain concentrations of iron (Fe) and zinc (Zn) ions. Phytic acid (PA) chelates these metal ions, reducing their bioavailability in the digestive tract. We present a high-throughput assay for quantification of PA and its application in screening a breeding population. After extraction in 96-well megatiter plates, PA content was determined from the phosphate released after treatment with a commercially available phytase enzyme. In a set of 330 breeding lines of wheat grown in the field over 3 years as part of a HarvestPlus breeding program for high grain Fe and Zn, our assay unraveled variation for PA that ranged from 0.90 to 1.72% with a mean of 1.24%. PA content was not associated with grain yield. High yielding lines were further screened for low molar PA/Fe and PA/Zn ratios for increased metal ion bioavailability, demonstrating the utility of our assay. Genome-wide association study revealed 21 genetic associations, six of which were consistent across years. Five of these associations mapped to chromosomes 1A, 2A, 2D, 5A, and 7D. Additivity over four of these haplotypes accounted for an ∼10% reduction in PA. Our study demonstrates it is possible to scale up assays to directly select for low grain PA in forward breeding programs.

Unraveling the Functional Role of NPF6 Transporters.

The nitrate transporter 1/peptide transporter (NPF) family represents a growing list of putative nitrate permeable transport proteins expressed within multiple cell types and tissues across a diverse range of plant species. Their designation as nitrate permeable and/or selective transporters is slowly being defined as more genes are characterized and their functional activities tested both in planta and in vitro. The most notable of the NPF family has been the Arabidopsis thaliana homolog, AtNPF6.3, previously known as AtNRT1.1 or CHL1. AtNPF6.3 has traditionally been characterized as a dual-affinity nitrate transporter contributing to root nitrate uptake in Arabidopsis. It has also been identified as a nitrate sensor which regulates the expression of high-affinity nitrate transport proteins NRT2s and lateral root development as a part of the primary nitrate response in plants. The sensor function of AtNPF6.3 has also been attributed to its auxin transport activity. Other homologs of AtNPF6.3 are now being described highlighting the variability in their functional capabilities (alternative substrates and kinetics) linking to structural aspects of the proteins. This review focusses on NPF6.3-like transport proteins and the knowledge that has been gained since their initial discovery over two decades ago. The review will investigate from a structural point of view how NPF6.3-like proteins may transport nitrate as well as other ions and what can be learned from structural uniqueness about predicted activities in plants.

Maize NPF6 Proteins Are Homologs of Arabidopsis CHL1 That Are Selective for Both Nitrate and Chloride.

Nitrate uptake by plant cells requires both high- and low-affinity transport activities. Arabidopsis thaliana nitrate transporter 1/peptide transporter family (NPF) 6.3 is a dual-affinity plasma membrane transport protein that has both high- and low-affinity functions. At-NPF6.3 imports and senses nitrate and is regulated by phosphorylation at Thr-101 (T101). A detailed functional analysis of two maize (Zea mays) homologs of At-NPF6.3 (Zm-NPF6.6 and Zm-NPF6.4) showed that Zm-NPF6.6 was a pH-dependent nonbiphasic high-affinity nitrate-specific transport protein. By contrast, maize NPF6.4 was a low-affinity nitrate transporter with efflux activity. When supplied chloride, NPF6.4 switched to a high-affinity chloride selective transporter, while NPF6.6 had only a low-affinity chloride transport activity. Structural predictions identified a nitrate binding His (H362) in NPF6.6 but not in NPF6.4. Mutation of NPF6.4 Tyr-370 to His (Y370H) resulted in saturable high-affinity nitrate transport activity and nitrate selectivity. Loss of H362 in NPF6.6 (H362Y) eliminated both nitrate and chloride transport. Furthermore, alterations to Thr-104, a conserved phosphorylation site in NPF6.6, resulted in a similar high-affinity nitrate transport activity with increased Km, whereas equivalent changes in NPF6.4 (T106) disrupted high-affinity chloride transport activity. NPF6 proteins exhibit different substrate specificity in plants and regulate nitrate transport affinity/selectivity using a conserved His residue.

Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago.

In legume-rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria.