PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is prevalent in upland rice and enhances root growth and hastens low phosphate signaling in wheat.

PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is a variably present gene that benefits crown root growth and phosphorus (P) sufficiency in rice (Oryza sativa). To explore the ecophysiological importance of this gene, we performed a biogeographic survey of landraces and cultivars, confirming that functional OsPSTOL1 alleles prevail in low nutrient and drought-prone rainfed ecosystems, whereas loss-of-function and absence haplotypes predominate in control-irrigated paddy varieties of east Asia. An evolutionary history analysis of OsPSTOL1 and related genes in cereal, determined it and other genes are kinase-only domain derivatives of membrane-associated receptor like kinases. Finally, to evaluate the potential value of this kinase of unknown function in another Gramineae, wheat (Triticum aestivum) lines overexpressing OsPSTOL1 were evaluated under field and controlled low P conditions. OsPSTOL1 enhances growth, crown root number, and overall root plasticity under low P in wheat. Survey of root and shoot crown transcriptomes at two developmental stages identifies transcription factors that are differentially regulated in OsPSTOL1 wheat that are similarly controlled by the gene in rice. In wheat, OsPSTOL1 alters the timing and amplitude of regulators of root development in dry soils and hastens induction of the core P-starvation response. OsPSTOL1 and related genes may aid more sustainable cultivation of cereal crops.

High-throughput 3D modelling to dissect the genetic control of leaf elongation in barley (Hordeum vulgare).

To optimize shoot growth and structure of cereals, we need to understand the genetic components controlling initiation and elongation. While measuring total shoot growth at high throughput using 2D imaging has progressed, recovering the 3D shoot structure of small grain cereals at a large scale is still challenging. Here, we present a method for measuring defined individual leaves of cereals, such as wheat and barley, using few images. Plant shoot modelling over time was used to measure the initiation and elongation of leaves in a bi-parental barley mapping population under low and high soil salinity. We detected quantitative trait loci (QTL) related to shoot growth per se, using both simple 2D total shoot measurements and our approach of measuring individual leaves. In addition, we detected QTL specific to leaf elongation and not to total shoot size. Of particular importance was the detection of a QTL on chromosome 3H specific to the early responses of leaf elongation to salt stress, a locus that could not be detected without the computer vision tools developed in this study.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in bread wheat.

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+ ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.

Translating knowledge about abiotic stress tolerance to breeding programmes.

Plant breeding and improvements in agronomic practice are making a consistent contribution to increasing global crop production year upon year. However, the rate of yield improvement currently lags behind the targets set to produce enough food to meet the demands of the predicted global population in 2050. Furthermore, crops that are exposed to harmful abiotic environmental factors (abiotic stresses, e.g. water limitation, salinity, extreme temperature) are prone to reduced yields. Here, we briefly describe the processes undertaken in conventional breeding programmes, which are usually designed to improve yields in near-optimal conditions rather than specifically breeding for improved crop yield stability under stressed conditions. While there is extensive fundamental research activity that examines mechanisms of plant stress tolerance, there are few examples that apply this research to improving commercial crop yields. There are notable exceptions, and we highlight some of these to demonstrate the magnitude of yield gains that could be made by translating agronomic, phenological and genetic solutions focused on improving or mitigating the effect of abiotic stress in the field; in particular, we focus on improvements in crop water-use efficiency and salinity tolerance. We speculate upon the reasons for the disconnect between research and research translation. We conclude that to realise untapped rapid gains towards food security targets new funding structures need to be embraced. Such funding needs to serve both the core and collaborative activities of the fundamental, pre-breeding and breeding research communities in order to expedite the translation of innovative research into the fields of primary producers.

Mapping of novel salt tolerance QTL in an Excalibur × Kukri doubled haploid wheat population.

KEY MESSAGE: Novel QTL for salinity tolerance traits have been detected using non-destructive and destructive phenotyping in bread wheat and were shown to be linked to improvements in yield in saline fields. Soil salinity is a major limitation to cereal production. Breeding new salt-tolerant cultivars has the potential to improve cereal crop yields. In this study, a doubled haploid bread wheat mapping population, derived from the bi-parental cross of Excalibur × Kukri, was grown in a glasshouse under control and salinity treatments and evaluated using high-throughput non-destructive imaging technology. Quantitative trait locus (QTL) analysis of this population detected multiple QTL under salt and control treatments. Of these, six QTL were detected in the salt treatment including one for maintenance of shoot growth under salinity (QG(1-5).asl-7A), one for leaf Na+ exclusion (QNa.asl-7A) and four for leaf K+ accumulation (QK.asl-2B.1, QK.asl-2B.2, QK.asl-5A and QK:Na.asl-6A). The beneficial allele for QG(1-5).asl-7A (the maintenance of shoot growth under salinity) was present in six out of 44 mainly Australian bread and durum wheat cultivars. The effect of each QTL allele on grain yield was tested in a range of salinity concentrations at three field sites across 2 years. In six out of nine field trials with different levels of salinity stress, lines with alleles for Na+ exclusion and/or K+ maintenance at three QTL (QNa.asl-7A, QK.asl-2B.2 and QK:Na.asl-6A) excluded more Na+ or accumulated more K+ compared to lines without these alleles. Importantly, the QK.asl-2B.2 allele for higher K+ accumulation was found to be associated with higher grain yield at all field sites. Several alleles at other QTL were associated with higher grain yields at selected field sites.

Identification of a Stelar-Localized Transport Protein That Facilitates Root-to-Shoot Transfer of Chloride in Arabidopsis.

Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl(-)) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl(-) xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl(-) efflux out of cells and was much less permeable to NO3(-). Shoot Cl(-) accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl(-) in plants, playing a role in the loading and the regulation of Cl(-) loading into the xylem of Arabidopsis roots during salinity stress.

SLAH1, a homologue of the slow type anion channel SLAC1, modulates shoot Cl- accumulation and salt tolerance in Arabidopsis thaliana.

Salinity tolerance is correlated with shoot chloride (Cl(-)) exclusion in multiple crops, but the molecular mechanisms of long-distance Cl(-) transport are poorly defined. Here, we characterize the in planta role of AtSLAH1 (a homologue of the slow type anion channel-associated 1 (SLAC1)). This protein, localized to the plasma membrane of root stelar cells, has its expression reduced by salt or ABA, which are key predictions for a protein involved with loading Cl(-) into the root xylem. Artificial microRNA knockdown mutants of AtSLAH1 had significantly reduced shoot Cl(-) accumulation when grown under low Cl(-), whereas shoot Cl(-) increased and the shoot nitrate/chloride ratio decreased following AtSLAH1 constitutive or stelar-specific overexpression when grown in high Cl(-) In both sets of overexpression lines a significant reduction in shoot biomass over the null segregants was observed under high Cl(-) supply, but not low Cl(-) supply. Further in planta data showed AtSLAH3 overexpression increased the shoot nitrate/chloride ratio, consistent with AtSLAH3 favouring nitrate transport. Heterologous expression of AtSLAH1 in Xenopus laevis oocytes led to no detectible transport, suggesting the need for post-translational modifications for AtSLAH1 to be active. Our in planta data are consistent with AtSLAH1 having a role in controlling root-to-shoot Cl(-) transport.

Evaluating contribution of ionic, osmotic and oxidative stress components towards salinity tolerance in barley.

BACKGROUND: Salinity tolerance is a physiologically multi-faceted trait attributed to multiple mechanisms. Three barley (Hordeum vulgare) varieties contrasting in their salinity tolerance were used to assess the relative contribution of ionic, osmotic and oxidative stress components towards overall salinity stress tolerance in this species, both at the whole-plant and cellular levels. In addition, transcriptional changes in the gene expression profile were studied for key genes mediating plant ionic and oxidative homeostasis (NHX; RBOH; SOD; AHA and GORK), to compare a contribution of transcriptional and post-translational factors towards the specific components of salinity tolerance. RESULTS: Our major findings are two-fold. First, plant tissue tolerance was a dominating component that has determined the overall plant responses to salinity, with root K(+) retention ability and reduced sensitivity to stress-induced hydroxyl radical production being the main contributing tolerance mechanisms. Second, it was not possible to infer which cultivars were salinity tolerant based solely on expression profiling of candidate genes at one specific time point. For the genes studied and the time point selected that transcriptional changes in the expression of these specific genes had a small role for barley's adaptive responses to salinity. CONCLUSIONS: For better tissue tolerance, sodium sequestration, K(+) retention and resistance to oxidative stress all appeared to be crucial. Because these traits are highly interrelated, it is suggested that a major progress in crop breeding for salinity tolerance can be achieved only if these complementary traits are targeted at the same time. This study also highlights the essentiality of post translational modifications in plant adaptive responses to salinity.

Expression of the Arabidopsis vacuolar H⁺-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field.

Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H⁺-PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high-throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse-grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to null segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mM NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild-type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse- or field-grown plants. This study validates our greenhouse-based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields.

HVP10 encoding V-PPase is a prime candidate for the barley HvNax3 sodium exclusion gene: evidence from fine mapping and expression analysis.

In cereals, a common salinity tolerance mechanism is to limit accumulation of Na(+) in the shoot. In a cross between the barley variety Barque-73 (Hordeum vulgare ssp. vulgare) and the accession CPI-71284 of wild barley (H. vulgare ssp. spontaneum), the HvNax3 locus on chromosome 7H was found to determine a ~10-25 % difference in leaf Na(+) accumulation in seedlings grown in saline hydroponics, with the beneficial exclusion trait originating from the wild parent. The Na(+) exclusion allele was also associated with a 13-21 % increase in shoot fresh weight. The HvNax3 locus was delimited to a 0.4 cM genetic interval, where it cosegregated with the HVP10 gene for vacuolar H(+)-pyrophosphatase (V-PPase). Sequencing revealed that the mapping parents encoded identical HVP10 proteins, but salinity-induced mRNA expression of HVP10 was higher in CPI-71284 than in Barque-73, in both roots and shoots. By contrast, the expression of several other genes predicted by comparative mapping to be located in the HvNax3 interval was similar in the two parent lines. Previous work demonstrated roles for V-PPase in ion transport and salinity tolerance. We therefore considered transcription levels of HVP10 to be a possible basis for variation in shoot Na(+) accumulation and biomass production controlled by the HvNax3 locus under saline conditions. Potential mechanisms linking HVP10 expression patterns to the observed phenotypes are discussed.

Rapid non-destructive method to phenotype stomatal traits.

BACKGROUND: Stomata are tiny pores on the leaf surface that are central to gas exchange. Stomatal number, size and aperture are key determinants of plant transpiration and photosynthesis, and variation in these traits can affect plant growth and productivity. Current methods to screen for stomatal phenotypes are tedious and not high throughput. This impedes research on stomatal biology and hinders efforts to develop resilient crops with optimised stomatal patterning. We have developed a rapid non-destructive method to phenotype stomatal traits in three crop species: wheat, rice and tomato. RESULTS: The method consists of two steps. The first is the non-destructive capture of images of the leaf surface from plants in their growing environment using a handheld microscope; a process that only takes a few seconds compared to minutes for other methods. The second is to analyse stomatal features using a machine learning model that automatically detects, counts and measures stomatal number, size and aperture. The accuracy of the machine learning model in detecting stomata ranged from 88 to 99%, depending on the species, with a high correlation between measures of number, size and aperture using the machine learning models and by measuring them manually. The rapid method was applied to quickly identify contrasting stomatal phenotypes. CONCLUSIONS: We developed a method that combines rapid non-destructive imaging of leaf surfaces with automated image analysis. The method provides accurate data on stomatal features while significantly reducing time for data acquisition and analysis. It can be readily used to phenotype stomata in large populations in the field and in controlled environments.

Corrigendum to: Identification of salt tolerance QTL in a wheat RIL mapping population using destructive and non-destructive phenotyping.

Bread wheat (Triticum aestivum L.) is one of the most important food crops, however it is only moderately tolerant to salinity stress. To improve wheat yield under saline conditions, breeding for improved salinity tolerance of wheat is needed. We have identified nine quantitative trail loci (QTL) for different salt tolerance sub-traits in a recombinant inbred line (RIL) population, derived from the bi-parental cross of Excalibur × Kukri. This population was screened for salinity tolerance subtraits using a combination of both destructive and non-destructive phenotyping. Genotyping by sequencing (GBS) was used to construct a high-density genetic linkage map, consisting of 3236 markers, and utilised for mapping QTL. Of the nine mapped QTL, six were detected under salt stress, including QTL for maintenance of shoot growth under salinity (QG ( 1-5 ) .asl -5A , QG ( 1-5 ) .asl -7B ) sodium accumulation (QNa.asl -2A ), chloride accumulation (QCl.asl -2A , QCl.asl -3A ) and potassium : sodium ratio (QK :Na.asl -2DS2 ). Potential candidate genes within these QTL intervals were shortlisted using bioinformatics tools. These findings are expected to facilitate the breeding of new salt tolerant wheat cultivars. Soil salinity causes major yield losses in bread wheat, which is moderately tolerant to salinity stress. Using high throughput genotyping and phenotyping techniques, we identified quantitative trail loci (QTL) for different salt tolerance sub-traits in bread wheat and shortlisted potential candidate genes. These QTL and candidate genes may prove useful in breeding for salt tolerant wheat cultivars.

Identification of salt tolerance QTL in a wheat RIL mapping population using destructive and non-destructive phenotyping.

Bread wheat (Triticum aestivum L.) is one of the most important food crops, however it is only moderately tolerant to salinity stress. To improve wheat yield under saline conditions, breeding for improved salinity tolerance of wheat is needed. We have identified nine quantitative trail loci (QTL) for different salt tolerance sub-traits in a recombinant inbred line (RIL) population, derived from the bi-parental cross of Excalibur × Kukri. This population was screened for salinity tolerance subtraits using a combination of both destructive and non-destructive phenotyping. Genotyping by sequencing (GBS) was used to construct a high-density genetic linkage map, consisting of 3236 markers, and utilised for mapping QTL. Of the nine mapped QTL, six were detected under salt stress, including QTL for maintenance of shoot growth under salinity (QG(1-5).asl-5A, QG(1-5).asl-7B) sodium accumulation (QNa.asl-2A), chloride accumulation (QCl.asl-2A, QCl.asl-3A) and potassium:sodium ratio (QK:Na.asl-2DS2). Potential candidate genes within these QTL intervals were shortlisted using bioinformatics tools. These findings are expected to facilitate the breeding of new salt tolerant wheat cultivars.

Identifying the genetic control of salinity tolerance in the bread wheat landrace Mocho de Espiga Branca.

Salinity tolerance in bread wheat is frequently reported to be associated with low leaf sodium (Na+) concentrations. However, the Portuguese landrace, Mocho de Espiga Branca, accumulates significantly higher leaf Na+ but has comparable salinity tolerance to commercial bread wheat cultivars. To determine the genetic loci associated with the salinity tolerance of this landrace, an F2 mapping population was developed by crossing Mocho de Espiga Branca with the Australian cultivar Gladius. The population was phenotyped for 19 salinity tolerance subtraits using both non-destructive and destructive techniques. Genotyping was performed using genotyping-by-sequencing (GBS). Genomic regions associated with salinity tolerance were detected on chromosomes 1A, 1D, 4B and 5A for the subtraits of relative and absolute growth rate (RGR, AGR respectively), and on chromosome 2A, 2B, 4D and 5D for Na+, potassium (K+) and chloride (Cl-) accumulation. Candidate genes that encode proteins associated with salinity tolerance were identified within the loci including Na+/H+ antiporters, K+ channels, H+-ATPase, calcineurin B-like proteins (CBLs), CBL-interacting protein kinases (CIPKs), calcium dependent protein kinases (CDPKs) and calcium-transporting ATPase. This study provides a new insight into the genetic control of salinity tolerance in a Na+ accumulating bread wheat to assist with the future development of salt tolerant cultivars.

Quantifying the three main components of salinity tolerance in cereals.

Salinity stress is a major factor inhibiting cereal yield throughout the world. Tolerance to salinity stress can be considered to contain three main components: Na(+) exclusion, tolerance to Na(+) in the tissues and osmotic tolerance. To date, most experimental work on salinity tolerance in cereals has focused on Na(+) exclusion due in part to its ease of measurement. It has become apparent, however, that Na(+) exclusion is not the sole mechanism for salinity tolerance in cereals, and research needs to expand to study osmotic tolerance and tissue tolerance. Here, we develop assays for high throughput quantification of Na(+) exclusion, Na(+) tissue tolerance and osmotic tolerance in 12 Triticum monococcum accessions, mainly using commercially available image capture and analysis equipment. We show that different lines use different combinations of the three tolerance mechanisms to increase their total salinity tolerance, with a positive correlation observed between a plant's total salinity tolerance and the sum of its proficiency in Na(+) exclusion, osmotic tolerance and tissue tolerance. The assays developed in this study can be easily adapted for other cereals and used in high throughput, forward genetic experiments to elucidate the molecular basis of these components of salinity tolerance.

AVP1: One Protein, Many Roles.

Constitutive expression of the Arabidopsis vacuolar proton-pumping pyrophosphatase (H+-PPase) gene (AVP1) increases plant growth under various abiotic stress conditions and, importantly, under nonstressed conditions. Many interpretations have been proposed to explain these phenotypes, including greater vacuolar ion sequestration, increased auxin transport, enhanced heterotrophic growth, and increased transport of sucrose from source to sink tissues. In this review, we evaluate all the roles proposed for AVP1, using findings published to date from mutant plants lacking functional AVP1 and transgenic plants expressing AVP1. It is clear that AVP1 is one protein with many roles, and that one or more of these roles act to enhance plant growth. The complexity suggests that a systems biology approach to evaluate biological networks is required to investigate these intertwined roles.

Expressing Arabidopsis thaliana V-ATPase subunit C in barley (Hordeum vulgare) improves plant performance under saline condition by enabling better osmotic adjustment.

Salinity is a global problem affecting agriculture that results in an estimated US$27 billion loss in revenue per year. Overexpression of vacuolar ATPase subunits has been shown to be beneficial in improving plant performance under saline conditions. Most studies, however, have not shown whether overexpression of genes encoding ATPase subunits results in improvements in grain yield, and have not investigated the physiological mechanisms behind the improvement in plant growth. In this study, we constitutively expressed Arabidopsis Vacuolar ATPase subunit C (AtVHA-C) in barley. Transgenic plants were assessed for agronomical and physiological characteristics, such as fresh and dry biomass, leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ concentration, when grown in either 0 or 300mM NaCl. When compared with non-transformed barley, AtVHA-C expressing barley lines had a smaller reduction in both biomass and grain yield under salinity stress. The transgenic lines accumulated Na+ and K+ in leaves for osmotic adjustment. This in turn saves energy consumed in the synthesis of organic osmolytes that otherwise would be needed for osmotic adjustment.

Variation in shoot tolerance mechanisms not related to ion toxicity in barley.

Soil salinity can severely reduce crop growth and yield. Many studies have investigated salinity tolerance mechanisms in cereals using phenotypes that are relatively easy to measure. The majority of these studies measured the accumulation of shoot Na+ and the effect this has on plant growth. However, plant growth is reduced immediately after exposure to NaCl before Na+ accumulates to toxic concentrations in the shoot. In this study, nondestructive and destructive measurements are used to evaluate the responses of 24 predominately Australian barley (Hordeum vulgare L.) lines at 0, 150 and 250mM NaCl. Considerable variation for shoot tolerance mechanisms not related to ion toxicity (shoot ion-independent tolerance) was found, with some lines being able to maintain substantial growth rates under salt stress, whereas others stopped growing. Hordeum vulgare spp. spontaneum accessions and barley landraces predominantly had the best shoot ion independent tolerance, although two commercial cultivars, Fathom and Skiff, also had high tolerance. The tolerance of cv. Fathom may be caused by a recent introgression from H. vulgare L. spp. spontaneum. This study shows that the most salt-tolerant barley lines are those that contain both shoot ion-independent tolerance and the ability to exclude Na+ from the shoot (and thus maintain high K+:Na+ ratios).