SOS1 tonoplast neo-localization and the RGG protein SALTY are important in the extreme salinity tolerance of Salicornia bigelovii.

The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na+ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.

The role of PQL genes in response to salinity tolerance in Arabidopsis and barley.

While soil salinity is a global problem, how salt enters plant root cells from the soil solution remains underexplored. Non-selective cation channels (NSCCs) are suggested to be the major pathway for the entry of sodium ions (Na+), yet their genetic constituents remain unknown. Yeast PQ loop (PQL) proteins were previously proposed to encode NSCCs, but the role of PQLs in plants is unknown. The hypothesis tested in this research is that PQL proteins constitute NSCCs mediating some of the Na+ influx into the root, contributing to ion accumulation and the inhibition of growth in saline conditions. We identified plant PQL homologues, and studied the role of one clade of PQL genes in Arabidopsis and barley. Using heterologous expression of AtPQL1a and HvPQL1 in HEK293 cells allowed us to resolve sizable inwardly directed currents permeable to monovalent cations such as Na+, K+, or Li+ upon membrane hyperpolarization. We observed that GFP-tagged PQL proteins localized to intracellular membrane structures, both when transiently over-expressed in tobacco leaf epidermis and in stable Arabidopsis transformants. Expression of AtPQL1a, AtPQL1b, and AtPQL1c was increased by salt stress in the shoot tissue compared to non-stressed plants. Mutant lines with altered expression of AtPQL1a, AtPQL1b, and AtPQL1c developed larger rosettes in saline conditions, while altered levels of AtPQL1a severely reduced development of lateral roots in all conditions. This study provides the first step toward understanding the function of PQL proteins in plants and the role of NSCC in salinity tolerance.

The genome of Chenopodium quinoa.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm.

BACKGROUND: Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g(-1) Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g(-1) Fe in endosperm. METHODOLOGY/PRINCIPAL FINDINGS: Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g(-1) Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g(-1) Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm. CONCLUSIONS: The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.

Rice plants expressing the moss sodium pumping ATPase PpENA1 maintain greater biomass production under salt stress.

High cytosolic concentrations of Na+ inhibit plant growth and development. To maintain low cytosolic concentrations of Na+ , higher plants use membrane-bound transporters that drive the efflux of Na+ or partition Na+ ions from the cytosol, either to the extracellular compartment or into the vacuole. Bryophytes also use an energy-dependent Na+ pumping ATPase, not found in higher plants, to efflux Na+ . To investigate whether this transporter can increase the salt tolerance of crop plants, Oryza sativa has been transformed with the Physcomitrella patens Na+ pumping ATPase (PpENA1). When grown in solutions containing 50 mm NaCl, plants constitutively expressing the PpENA1 gene are more salt tolerant and produce greater biomass than controls. Transgenics and controls accumulate similar amounts of Na+ in leaf and root tissues under stress, which indicates that the observed tolerance is not because of Na+ exclusion. Moreover, inductively coupled plasma analysis reveals that the concentration of other ions in the transformants and the controls is similar. The transgenic lines are developmentally normal and fertile, and the transgene expression levels remain stable in subsequent generations. GFP reporter fusions, which do not alter the ability of PpENA1 to complement a salt-sensitive yeast mutant, indicate that when it is expressed in plant tissues, the PpENA1 protein is located in the plasma membrane. PpENA1 peptides are found in plasma membrane fractions supporting the plasma membrane targeting. The results of this study demonstrate the utility of PpENA1 as a potential tool for engineering salinity tolerance in important crop species.

Structural and functional analyses of PpENA1 provide insights into cation binding by type IID P-type ATPases in lower plants and fungi.

PpENA1 is a membrane-spanning transporter from the moss Physcomitrella patens, and is the first type IID P-type ATPase to be reported in the plant kingdom. In Physcomitrella, PpENA1 is essential for normal growth under moderate salt stress, while in yeast, type IID ATPases provide a vital efflux mechanism for cells under high salt conditions by selectively transporting Na+ or K+ across the plasma membrane. To investigate the structural basis for cation-binding within the type IID ATPase subfamily, we used homology modeling to identify a highly conserved cation-binding pocket between membrane helix (MH) 4 and MH 6 of the membrane-spanning pore of PpENA1. Mutation of specific charged and polar residues on MHs 4-6 resulted in a decrease or loss of protein activity as measured by complementation assays in yeast. The E298S mutation on MH 4 of PpENA1 had the most significant effect on activity despite the presence of a serine at this position in fungal type IID ATPases. Activity was partially restored in an inactivated PpENA1 mutant by the insertion of two additional serine residues on MH 4 and one on MH 6 based on the presence of these residues in fungal type IID ATPases. Our results suggest that the residues responsible for cation-binding in PpENA1 are distinct from those in fungal type IID ATPases, and that a fungal-type cation binding site can be successfully engineered into the moss protein.

Exclusion of Na+ via sodium ATPase (PpENA1) ensures normal growth of Physcomitrella patens under moderate salt stress.

The bryophyte Physcomitrella patens is unlike any other plant identified to date in that it possesses a gene that encodes an ENA-type Na(+)-ATPase. To complement previous work in yeast (Saccharomyces cerevisiae), we determined the importance of having a Na(+)-ATPase in planta by conducting physiological analyses of PpENA1 in Physcomitrella. Expression studies showed that PpENA1 is up-regulated by NaCl and, to a lesser degree, by osmotic stress. Maximal induction is obtained after 8 h at 60 mm NaCl or above. No other abiotic stress tested led to significant increases in PpENA1 expression. In the gametophyte, strong expression was confined to the rhizoids, stem, and the basal part of the leaf. In the protonemata, expression was ubiquitous with a few filaments showing stronger expression. At 100 mm NaCl, wild-type plants were able to maintain a higher K(+)-to-Na(+) ratio than the PpENA1 (ena1) knockout gene, but at higher NaCl concentrations no difference was observed. Although no difference in chlorophyll content was observed between ena1 and wild type at 100 mm NaCl, the impaired Na(+) exclusion in ena1 plants led to an approximately 40% decrease in growth.

HKT1;5-like cation transporters linked to Na+ exclusion loci in wheat, Nax2 and Kna1.

Bread wheat (Triticum aestivum) has a greater ability to exclude Na+ from its leaves and is more salt tolerant than durum wheat (Triticum turgidum L. subsp. durum [Desf.]). A novel durum wheat, Line 149, was found to contain a major gene for Na+ exclusion, Nax2, which removes Na+ from the xylem in the roots and leads to a high K+-to-Na+ ratio in the leaves. Nax2 was mapped to the distal region on chromosome 5AL based on linkage to microsatellite markers. The Nax2 locus on 5AL coincides with the locus for a putative Na+ transporter, HKT1;5 (HKT8). The Nax2 region on 5AL is homoeologous to the region on chromosome 4DL containing the major Na+ exclusion locus in bread wheat, Kna1. A gene member of the HKT1;5 family colocates to the deletion bin containing Kna1 on chromosome 4DL. This work provides evidence that Nax2 and Kna1 are strongly associated with HKT1;5 genes.