Sensor-based phenotyping of above-ground plant-pathogen interactions.

Plant pathogens cause yield losses in crops worldwide. Breeding for improved disease resistance and management by precision agriculture are two approaches to limit such yield losses. Both rely on detecting and quantifying signs and symptoms of plant disease. To achieve this, the field of plant phenotyping makes use of non-invasive sensor technology. Compared to invasive methods, this can offer improved throughput and allow for repeated measurements on living plants. Abiotic stress responses and yield components have been successfully measured with phenotyping technologies, whereas phenotyping methods for biotic stresses are less developed, despite the relevance of plant disease in crop production. The interactions between plants and pathogens can lead to a variety of signs (when the pathogen itself can be detected) and diverse symptoms (detectable responses of the plant). Here, we review the strengths and weaknesses of a broad range of sensor technologies that are being used for sensing of signs and symptoms on plant shoots, including monochrome, RGB, hyperspectral, fluorescence, chlorophyll fluorescence and thermal sensors, as well as Raman spectroscopy, X-ray computed tomography, and optical coherence tomography. We argue that choosing and combining appropriate sensors for each plant-pathosystem and measuring with sufficient spatial resolution can enable specific and accurate measurements of above-ground signs and symptoms of plant disease.

Proton-pumping pyrophosphatase homeolog expression is a dynamic trait in bread wheat (Triticum aestivum).

Proton-pumping pyrophosphatases (H+-PPases) have been shown to enhance biomass and yield. However, to date, there has been little work towards identify genes encoding H+-PPases in bread wheat (Triticum aestivum) (TaVPs) and limited knowledge on how the expression of these genes varies across different growth stages and tissue types. In this study, the IWGSC database was used to identify two novel TaVP genes, TaVP4 and TaVP5, and elucidate the complete homeolog sequences of the three known TaVP genes, bringing the total number of bread wheat TaVPs from 9 to 15. Gene expression levels of each TaVP homeolog were assessed using quantitative real-time PCR (qRT-PCR) in four diverse wheat varieties in terms of phenotypic traits related to high vacuolar pyrophosphatase expression. Homeolog expression was analyzed across multiple tissue types and developmental stages. Expression levels of the TaVP homeologs were found to vary significantly between varieties, tissues and plant developmental stages. During early development (Z10 and Z13), expressions of TaVP1 and TaVP2 homeologs were higher in shoot tissue than root tissue, with both shoot and root expression increasing in later developmental stages (Z22). TaVP2-D was expressed in all varieties and tissue types and was the most highly expressed homeolog at all developmental stages. Expression of the TaVP3 homeologs was restricted to developing grain (Z75), while TaVP4 homeolog expression was higher at Z22 than earlier developmental stages. Variation in TaVP4B was detected among varieties at Z22 and Z75, with Buck Atlantico (high biomass) and Scout (elite Australian cultivar) having the highest levels of expression. These findings offer a comprehensive overview of the bread wheat H+-PPase family and identify variation in TaVP homeolog expression that will be of use to improve the growth, yield, and abiotic stress tolerance of bread wheat.

The phosphoproteome of rice leaves responds to water and nitrogen supply.

The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants employ complex systems for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, in plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic stress events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

Continuous monitoring of plant sodium transport dynamics using clinical PET.

BACKGROUND: The absorption, translocation, accumulation and excretion of substances are fundamental processes in all organisms including plants, and have been successfully studied using radiotracers labelled with 11C, 13N, 14C and 22Na since 1939. Sodium is one of the most damaging ions to the growth and productivity of crops. Due to the significance of understanding sodium transport in plants, a significant number of studies have been carried out to examine sodium influx, compartmentation, and efflux using 22Na- or 24Na-labeled salts. Notably, however, most of these studies employed destructive methods, which has limited our understanding of sodium flux and distribution characteristics in real time, in live plants. Positron emission tomography (PET) has been used successfully in medical research and diagnosis for decades. Due to its ability to visualise and assess physiological and metabolic function, PET imaging has also begun to be employed in plant research. Here, we report the use of a clinical PET scanner with a 22Na tracer to examine 22Na-influx dynamics in barley plants (Hordeum vulgare L. spp. Vulgare-cultivar Bass) under variable nutrient levels, alterations in the day/night light cycle, and the presence of sodium channel inhibitors. RESULTS: 3D dynamic PET images of whole plants show readily visible 22Na translocation from roots to shoots in each examined plant, with rates influenced by both nutrient status and channel inhibition. PET images show that plants cultivated in low-nutrient media transport more 22Na than plants cultivated in high-nutrient media, and that 22Na uptake is suppressed in the presence of a cation-channel inhibitor. A distinct diurnal pattern of 22Na influx was discernible in curves displaying rates of change of relative radioactivity. Plants were found to absorb more 22Na during the light period, and anticipate the change in the light/dark cycle by adjusting the sodium influx rate downward in the dark period, an effect not previously described experimentally. CONCLUSIONS: We demonstrate the utility of clinical PET/CT scanners for real-time monitoring of the temporal dynamics of sodium transport in plants. The effects of nutrient deprivation and of ion channel inhibition on sodium influx into barley plants are shown in two proof-of-concept experiments, along with the first-ever 3D-imaging of the light and dark sodium uptake cycles in plants. This method carries significant potential for plant biology research and, in particular, in the context of genetic and treatment effects on sodium acquisition and toxicity in plants.

Improved Yield and Photosynthate Partitioning in AVP1 Expressing Wheat (Triticum aestivum) Plants.

A fundamental factor to improve crop productivity involves the optimization of reduced carbon translocation from source to sink tissues. Here, we present data consistent with the positive effect that the expression of the Arabidopsis thaliana H+-PPase (AVP1) has on reduced carbon partitioning and yield increases in wheat. Immunohistochemical localization of H+-PPases (TaVP) in spring wheat Bobwhite L. revealed the presence of this conserved enzyme in wheat vasculature and sink tissues. Of note, immunogold imaging showed a plasma membrane localization of TaVP in sieve element- companion cell complexes of Bobwhite source leaves. These data together with the distribution patterns of a fluorescent tracer and [U14C]-sucrose are consistent with an apoplasmic phloem-loading model in wheat. Interestingly, 14C-labeling experiments provided evidence for enhanced carbon partitioning between shoots and roots, and between flag leaves and milk stage kernels in AVP1 expressing Bobwhite lines. In keeping, there is a significant yield improvement triggered by the expression of AVP1 in these lines. Green house and field grown transgenic wheat expressing AVP1 also produced higher grain yield and number of seeds per plant, and exhibited an increase in root biomass when compared to null segregants. Another agriculturally desirable phenotype showed by AVP1 Bobwhite plants is a robust establishment of seedlings.

The intersection of nitrogen nutrition and water use in plants: new paths toward improved crop productivity.

Water and nitrogen availability limit crop productivity globally more than most other environmental factors. Plant availability of macronutrients such as nitrate is, to a large extent, regulated by the amount of water available in the soil, and, during drought episodes, crops can become simultaneously water and nitrogen limited. In this review, we explore the intricate relationship between water and nitrogen transport in plants, from transpiration-driven mass flow in the soil to uptake by roots via membrane transporters and channels and transport to aerial organs. We discuss the roles of root architecture and of suberized hydrophobic root barriers governing apoplastic water and nitrogen movement into the vascular system. We also highlight the need to identify the signalling cascades regulating water and nitrogen transport, as well as the need for targeted physiological analyses of plant traits influencing water and nitrogen uptake. We further advocate for incorporation of new phenotyping technologies, breeding strategies, and agronomic practices to improve crop yield in water- and nitrogen-limited production systems.

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.

Plasma-membrane electrical responses to salt and osmotic gradients contradict radiotracer kinetics, and reveal Na+-transport dynamics in rice (Oryza sativa L.).

MAIN CONCLUSION: A systematic analysis of NaCl-dependent, plasma-membrane depolarization (∆∆Ψ) in rice roots calls into question the current leading model of rapid membrane cycling of Na+ under salt stress. To investigate the character and mechanisms of Na+ influx into roots, Na+-dependent changes in plasma-membrane electrical potentials (∆∆Ψ) were measured in root cells of intact rice (Oryza sativa L., cv. Pokkali) seedlings. As external sodium concentrations ([Na+]ext) were increased in a step gradient from 0 to 100 mM, membrane potentials depolarized in a saturable manner, fitting a Michaelis-Menten model and contradicting the linear (non-saturating) models developed from radiotracer studies. Clear differences in saturation patterns were found between plants grown under low- and high-nutrient (LN and HN) conditions, with LN plants showing greater depolarization and higher affinity for Na+ (i.e., higher Vmax and lower Km) than HN plants. In addition, counterion effects on ∆∆Ψ were pronounced in LN plants (with ∆∆Ψ decreasing in the order: Cl- > SO42- > HPO 4 2- ), but not seen in HN plants. When effects of osmotic strength, Cl- influx, K+ efflux, and H+-ATPase activity on ∆∆Ψ were accounted for, resultant Km and Vmax values suggested that a single, dominant Na+-transport mechanism was operating under each nutritional condition, with Km values of 1.2 and 16 mM for LN and HN plants, respectively. Comparing saturating patterns of depolarization to linear patterns of 24Na+ radiotracer influx leads to the conclusion that electrophysiological and tracer methods do not report the same phenomena and that the current model of rapid transmembrane sodium cycling may require revision.

Nitrate uptake and its regulation in relation to improving nitrogen use efficiency in cereals.

On average less than half of the applied N is captured by crops, thus there is scope and need to improve N uptake in cereals. With nitrate (NO3-) being the main form of N available to cereal crops there has been a significant global research effort to understand plant NO3- uptake. Despite this, our knowledge of the NO3- uptake system is not sufficient to easily target ways to improve NO3- uptake. Based on this there is an identified need to better understand the NO3- uptake system and the signalling molecules that modulate it. With strong transcriptional control governing the NO3- uptake system, we also need new leads for modulating transcription of NO3- transporter genes.

Integrated genomics, physiology and breeding approaches for improving nitrogen use efficiency in potato: translating knowledge from other crops.

Potato plays a key role in global food and nutritional security. Potato is an N fertiliser-responsive crop, producing high tuber yields. However, excessive use of N can result in environmental damage and high production costs, hence improving nitrogen use efficiency (NUE) of potato plants is one of the sustainable options to address these issues and increase yield. Advanced efforts have been undertaken to improve NUE in other plants like Arabidopsis, rice, wheat and maize through molecular and physiological approaches. Conversely, in potato, NUE studies have predominantly focussed on agronomy or soil management, except for a few researchers who have measured gene expression and proteins relevant to N uptake or metabolism. The focus of this review is to adapt knowledge gained from other plants to inform investigation of N metabolism and associated traits in potato with the aim of improving potato NUE using integrated genomics, physiology and breeding methods.

Structural variations in wheat HKT1;5 underpin differences in Na+ transport capacity.

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na+) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na+-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K m) for the Na+ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D471/a gap and D474/G473 that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na+ exclusion in wheat that leads to an improved salinity tolerance in the field.

Transition from a maternal to external nitrogen source in maize seedlings.

Maximizing NO3- uptake during seedling development is important as it has a major influence on plant growth and yield. However, little is known about the processes leading to, and involved in, the initiation of root NO3- uptake capacity in developing seedlings. This study examines the physiological processes involved in root NO3- uptake and metabolism, to gain an understanding of how the NO3- uptake system responds to meet demand as maize seedlings transition from seed N use to external N capture. The concentrations of seed-derived free amino acids within root and shoot tissues are initially high, but decrease rapidly until stabilizing eight days after imbibition (DAI). Similarly, shoot N% decreases, but does not stabilize until 12-13 DAI. Following the decrease in free amino acid concentrations, root NO3- uptake capacity increases until shoot N% stabilizes. The increase in root NO3- uptake capacity corresponds with a rapid rise in transcript levels of putative NO3- transporters, ZmNRT2.1 and ZmNRT2.2. The processes underlying the increase in root NO3- uptake capacity to meet N demand provide an insight into the processes controlling N uptake.

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.

Nitrogen assimilation system in maize is regulated by developmental and tissue-specific mechanisms.

KEY MESSAGE: We found metabolites, enzyme activities and enzyme transcript abundances vary significantly across the maize lifecycle, but weak correlation exists between the three groups. We identified putative genes regulating nitrate assimilation. Progress in improving nitrogen (N) use efficiency (NUE) of crop plants has been hampered by the complexity of the N uptake and utilisation systems. To understand this complexity we measured the activities of seven enzymes and ten metabolites related to N metabolism in the leaf and root tissues of Gaspe Flint maize plants grown in 0.5 or 2.5 mM NO3 (-) throughout the lifecycle. The amino acids had remarkably similar profiles across the lifecycle except for transient responses, which only appeared in the leaves for aspartate or in the roots for asparagine, serine and glycine. The activities of the enzymes for N assimilation were also coordinated to a certain degree, most noticeably with a peak in root activity late in the lifecycle, but with wide variation in the activity levels over the course of development. We analysed the transcriptional data for gene sets encoding the measured enzymes and found that, unlike the enzyme activities, transcript levels of the corresponding genes did not exhibit the same coordination across the lifecycle and were only weakly correlated with the levels of various amino acids or individual enzyme activities. We identified gene sets which were correlated with the enzyme activity profiles, including seven genes located within previously known quantitative trait loci for enzyme activities and hypothesise that these genes are important for the regulation of enzyme activities. This work provides insights into the complexity of the N assimilation system throughout development and identifies candidate regulatory genes, which warrant further investigation in efforts to improve NUE in crop plants.

Maize maintains growth in response to decreased nitrate supply through a highly dynamic and developmental stage-specific transcriptional response.

Elucidation of the gene networks underlying the response to N supply and demand will facilitate the improvement of the N uptake efficiency of plants. We undertook a transcriptomic analysis of maize to identify genes responding to both a non-growth-limiting decrease in NO3- provision and to development-based N demand changes at seven representative points across the life cycle. Gene co-expression networks were derived by cluster analysis of the transcript profiles. The majority of NO3--responsive transcription occurred at 11 (D11), 18 (D18) and 29 (D29) days after emergence, with differential expression predominating in the root at D11 and D29 and in the leaf at D18. A cluster of 98 probe sets was identified, the expression pattern of which is similar to that of the high-affinity NO3- transporter (NRT2) genes across the life cycle. The cluster is enriched with genes encoding enzymes and proteins of lipid metabolism and transport, respectively. These are candidate genes for the response of maize to N supply and demand. Only a few patterns of differential gene expression were observed over the entire life cycle; however, the composition of the classes of the genes differentially regulated at individual time points was unique, suggesting tightly controlled regulation of NO3--responsive gene expression.

Variation for N Uptake System in Maize: Genotypic Response to N Supply.

An understanding of the adaptations made by plants in their nitrogen (N) uptake systems in response to reduced N supply is important to the development of cereals with enhanced N uptake efficiency (NUpE). Twenty seven diverse genotypes of maize (Zea mays, L.) were grown in hydroponics for 3 weeks with limiting or adequate N supply. Genotypic response to N was assessed on the basis of biomass characteristics and the activities of the nitrate ([Formula: see text]) and ammonium ([Formula: see text]) high-affinity transport systems. Genotypes differed greatly for the ability to maintain biomass with reduced N. Although, the N response in underlying biomass and N transport related characteristics was less than that for biomass, there were clear relationships, most importantly, lines that maintained biomass at reduced N maintained net N uptake with no change in size of the root relative to the shoot. The root uptake capacity for both [Formula: see text] and [Formula: see text] increased with reduced N. Transcript levels of putative [Formula: see text] and [Formula: see text] transporter genes in the root tissue of a subset of the genotypes revealed that predominately ZmNRT2 transcript levels responded to N treatments. The correlation between the ratio of transcripts of ZmNRT2.2 between the two N levels and a genotype's ability to maintain biomass with reduced N suggests a role for these transporters in enhancing NUpE. The observed variation in the ability to capture N at low N provides scope for both improving NUpE in maize and also to better understand the N uptake system in cereals.

Genetic approaches to enhancing nitrogen-use efficiency (NUE) in cereals: challenges and future directions.

Over 100million tonnes of nitrogen (N) fertiliser are applied globally each year to maintain high yields in agricultural crops. The rising price of N fertilisers has made them a major cost for farmers. Inefficient use of N fertiliser leads to substantial environmental problems through contamination of air and water resources and can be a significant economic cost. Consequently, there is considerable need to improve the way N fertiliser is used in farming systems. The efficiency with which crops use applied N fertiliser - the nitrogen-use efficiency (NUE) - is currently quite low for cereals. This is the case in both high yielding environments and lower yielding environments characteristic of cereal growing regions of Australia. Multiple studies have attempted to identify the genetic basis of NUE, but the utility of the results is limited because of the complex nature of the trait and the magnitude of genotype by environment interaction. Transgenic approaches have been applied to improve plant NUE but with limited success, due, in part, to a combination of the complexity of the trait but also due to lack of accurate phenotyping methods. This review documents these two approaches and suggests future directions in improving cereal NUE with a focus on the Australian cereal industry.

The Na(+) transporter, TaHKT1;5-D, limits shoot Na(+) accumulation in bread wheat.

Bread wheat (Triticum aestivum L.) has a major salt tolerance locus, Kna1, responsible for the maintenance of a high cytosolic K(+) /Na(+) ratio in the leaves of salt stressed plants. The Kna1 locus encompasses a large DNA fragment, the distal 14% of chromosome 4DL. Limited recombination has been observed at this locus making it difficult to map genetically and identify the causal gene. Here, we decipher the function of TaHKT1;5-D, a candidate gene underlying the Kna1 locus. Transport studies using the heterologous expression systems Saccharomyces cerevisiae and Xenopus laevis oocytes indicated that TaHKT1;5-D is a Na(+) -selective transporter. Transient expression in Arabidopsis thaliana mesophyll protoplasts and in situ polymerase chain reaction indicated that TaHKT1;5-D is localised on the plasma membrane in the wheat root stele. RNA interference-induced silencing decreased the expression of TaHKT1;5-D in transgenic bread wheat lines which led to an increase in the Na(+) concentration in the leaves. This indicates that TaHKT1;5-D retrieves Na(+) from the xylem vessels in the root and has an important role in restricting the transport of Na(+) from the root to the leaves in bread wheat. Thus, TaHKT1;5-D confers the essential salinity tolerance mechanism in bread wheat associated with the Kna1 locus via shoot Na(+) exclusion and is critical in maintaining a high K(+) /Na(+) ratio in the leaves. These findings show there is potential to increase the salinity tolerance of bread wheat by manipulation of HKT1;5 genes.

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.