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Spinach (Spinacia oleracea L.) is grown extensively across the globe but requires irrigation to maintain both yield and leaf quality. Water resources are being put under increasing pressure as we endeavour to produce enough food, fuel and fibre for a growing human population. Thus, improving our water use efficiency is becoming ever more important. Here, we report the impacts of excess and deficit irrigation on spinach yield and quality. Mild deficit irrigation (-18% of commercial water application) had no significant impact on crop quality indicators or yield, with a trend for improved shelf life, when compared to the commercially irrigated crop. Alongside this, yield was only improved when water was applied in 40% above commercial levels. The yield and quality maintenance observed under mild water deficit was associated with a reduced stomatal index suggesting a useful trait for future breeding for drought tolerance. If this relationship is robust across environments and crop varieties, major water savings will be possible across the leafy salad agricultural sector, reducing the water footprint and improving the sustainability of crop production. Furthermore, this research has elucidated links between plant responses to irrigation modifications and the thermal spectra of the canopy, with the canopy range of temperature being the most sensitive indicator of soil moisture. There is potential for these relationships to be further developed across seasons and crops for use in future irrigation decision-making.

Highlights • Salad leaves lost 35–86% of their ascorbate during 10 d storage at 4 °C. • Water-washing of spinach leaves and leaf discs increased ascorbate loss. • Washing in presence of hypochlorite did not significantly increase ascorbate loss. • Mechanical agitation of spinach leaves during washing exacerbated ascorbate loss. • Oxalate was the major [14C]ascorbate by-product, indicating oxidative stress., Post-harvest treatments of pre-packaged salad leaves potentially cause l-ascorbate loss, but the mechanisms of ascorbate degradation remain incompletely understood, especially in planta. We explored the extent and pathways of ascorbate loss in variously washed and stored salad leaves. Ascorbate was assayed by 2,6-dichlorophenolindophenol titration, and pathways were monitored by 14C-radiolabelling followed by high-voltage electrophoresis. All leaves tested showed ascorbate loss during storage: lettuce showed the greatest percentage loss, wild rocket the least. Spinach leaves were particularly prone to losing ascorbate during washing, especially with simultaneous mechanical agitation; however, washing in the presence of hypochlorite did not significantly increase ascorbate loss. In spinach, [14C]oxalate was the major product of [14C]ascorbate degradation, suggesting that commercial washing causes oxidative stress. This study highlights that ascorbate/dehydroascorbic acid are lost via the oxidative pathway during washing and post-harvest storage of salad leaves. Thus changes to washing procedures could potentially increase the post-harvest retention of ascorbate.

Watercress (Rorippa nasturtium-aquaticum), wild rocket (Diplotaxis tenuifolia), and spinach (Spinacia oleracea) are commercial crops reported to have high concentrations of antioxidants, possibly contributing to disease prevention following human consumption. Following analysis of supermarket-purchased salad leaves, we report the antioxidant content potential of these species using two comparable techniques assessing the consistency between the assays – by the ferric reducing antioxidant power (FRAP) assay and the oxygen radical absorbance capacity (ORAC) assay. The leaves were harvested from both conventionally and organically managed crops, to investigate whether organic agriculture results in improved crop quality. Watercress had the highest FRAP and ability to scavenge free radicals, followed by spinach and rocket. For watercress and rocket, there was no significant effect of organic agriculture on FRAP and ORAC, but for spinach, the antioxidant potential was reduced and this was significant at the 5% level of probability for FRAP but not ORAC, although the trend was clear in both tests. We conclude that there is variation in salad crop antioxidant potential and that FRAP and ORAC are useful techniques for measuring antioxidants in these salad crops with similar ranking for each salad crop studied.

Background: Consuming watercress is thought to provide health benefits as a consequence of its phytonutrient composition. However, for watercress there are currently limited genetic resources underpinning breeding efforts for either yield or phytonutritional traits. In this paper, we use RNASeq data from twelve watercress accessions to characterize the transcriptome, perform candidate gene mining and conduct differential expression analysis for two key phytonutritional traits: antioxidant (AO) capacity and glucosinolate (GLS) content. Results: The watercress transcriptome was assembled to 80,800 transcripts (48,732 unigenes); 71 % of which were annotated based on orthology to Arabidopsis. Differential expression analysis comparing watercress accessions with ‘high’ and ‘low’ AO and GLS resulted in 145 and 94 differentially expressed loci for AO capacity and GLS respectively. Differentially expressed loci between high and low AO watercress were significantly enriched for genes involved in plant defence and response to stimuli, in line with the observation that AO are involved in plant stress-response. Differential expression between the high and low GLS watercress identified links to GLS regulation and also novel transcripts warranting further investigation. Additionally, we successfully identified watercress orthologs for Arabidopsis phenylpropanoid, GLS and shikimate biosynthesis pathway genes, and compiled a catalogue of polymorphic markers for future applications. Conclusions: Our work describes the first transcriptome of watercress and establishes the foundation for further molecular study by providing valuable resources, including sequence data, annotated transcripts, candidate genes and markers.

Background Consuming watercress is thought to provide health benefits as a consequence of its phytonutrient composition. However, for watercress there are currently limited genetic resources underpinning breeding efforts for either yield or phytonutritional traits. In this paper, we use RNASeq data from twelve watercress accessions to characterize the transcriptome, perform candidate gene mining and conduct differential expression analysis for two key phytonutritional traits: antioxidant (AO) capacity and glucosinolate (GLS) content. Results The watercress transcriptome was assembled to 80,800 transcripts (48,732 unigenes); 71 % of which were annotated based on orthology to Arabidopsis. Differential expression analysis comparing watercress accessions with ‘high’ and ‘low’ AO and GLS resulted in 145 and 94 differentially expressed loci for AO capacity and GLS respectively. Differentially expressed loci between high and low AO watercress were significantly enriched for genes involved in plant defence and response to stimuli, in line with the observation that AO are involved in plant stress-response. Differential expression between the high and low GLS watercress identified links to GLS regulation and also novel transcripts warranting further investigation. Additionally, we successfully identified watercress orthologs for Arabidopsis phenylpropanoid, GLS and shikimate biosynthesis pathway genes, and compiled a catalogue of polymorphic markers for future applications. Conclusions Our work describes the first transcriptome of watercress and establishes the foundation for further molecular study by providing valuable resources, including sequence data, annotated transcripts, candidate genes and markers. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2704-4) contains supplementary material, which is available to authorized users.

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Baby leaf salads are gaining in popularity over traditional whole head lettuce salads in response to consumer demand for greater variety and convenience in their diet. Baby lettuce leaves are mixed, washed and packaged as whole leaves, with a shelf-life of approximately 10 days post-processing. End of shelf-life, as determined by the consumer, is typified by bruising, water-logging and blackening of the leaves, but the biological events causing this phenotype have not been studied to date. We investigated the physiological and ultrastructural characteristics during postharvest shelf-life of two lettuce varieties with very different leaf morphologies. Membrane disruption was an important determinant of cell death in both varieties, although the timing and characteristics of breakdown was different in each with Lollo rossa showing signs of aging such as thylakoid disruption and plastoglobuli accumulation earlier than Cos. Membranes in Lollo rossa showed a later, but more distinct increase in permeability than in Cos, as indicated by electrolyte leakage and the presence of cytoplasmic fragments in the vacuole, but Cos membranes show distinct fractures towards the end of shelf-life. The tissue lost less than 25% fresh weight during shelf-life and there was little protein loss compared to developmentally aging leaves in an ambient environment. Biophysical measurements showed that breakstrength was significantly reduced in Lollo rossa, whereas irreversible leaf plasticity was significantly reduced in Cos leaves. The reversible elastic properties of both varieties changed throughout shelf-life. We compared the characteristics of shelf-life in both varieties of bagged lettuce leaves with other leafy salad crops and discuss the potential targets for future work to improve postharvest quality of baby leaf lettuce.

Developmental and biophysical leaf characteristics that influence post-harvest shelf life in lettuce, an important leafy crop, have been examined. The traits were studied using 60 informative F9 recombinant inbreed lines (RILs) derived from a cross between cultivated lettuce (Lactuca sativa cv. Salinas) and wild lettuce (L. serriola acc. UC96US23). Quantitative Trait Loci (QTL) for shelf life co-located most closely with those for leaf biophysical properties such as plasticity, elasticity and breakstrength suggesting that these are appropriate targets for molecular breeding for improved shelf life. Significant correlations were found between shelf life with leaf size, leaf weight, leaf chlorophyll content, leaf stomatal index and epidermal cell number per leaf, indicating that these pre-harvest leaf development traits confer postharvest properties. By studying the population in two contrasting environments in northern and southern Europe, the genotype by environment interaction effects of the QTL relevant to leaf development and shelf life were assessed. In total 107 QTL, distributed on all nine linkage groups, were detected from the 29 traits. Only five QTL were common in both environments. Several areas where many QTL co-located (hotspots) on the genome were identified, with relatively little overlap between developmental hotspots and those relating to shelf life. However, QTL for leaf biophysical properties (breakstrength, plasticity and elasticity) and cell area correlated well with shelf life, confirming that the ideal ideotype lettuce should have small cells with strong cell walls. The identification of QTL for leaf development, strength and longevity will lead to a better understanding of processability at a genetic and cellular level, and allow the improvement of salad leaf quality through marker assisted breeding.

Watercress (Nasturtium officinale R. Br.) is a nutrient intense, leafy crop that is consumed raw or in soups across the globe, but for which, currently no genomic resources or breeding programme exists. Promising morphological, biochemical and functional genomic variation was identified for the first time in a newly established watercress germplasm collection, consisting of 48 watercress accessions sourced from contrasting global locations. Stem length, stem diameter and anti-oxidant (AO) potential varied across the accessions. This variation was used to identify three extreme contrasting accessions for further analysis. Variation in global gene expression was investigated using an Affymetrix Arabidopsis ATH1 microarray gene chip, using the commercial control (C), an accession selected for dwarf phenotype with a high AO potential (dwarfAO, called ‘Boldrewood’) and one with high AO potential alone. A set of transcripts significantly differentially expressed between these three accessions, were identified, including transcripts involved in the regulation of growth and development and those involved in secondary metabolism. In particular, when differential gene expression was compared between C and dwarfAO, the dwarfAO was characterised by increased expression of genes encoding glucosinolates, which are known precursors of phenethyl isothiocyanate, linked to the anti-carcinogenic effects well-documented in watercress. This study provides the first analysis of natural variation across the watercress genome and has identified important underpinning information for future breeding for enhanced anti-carcinogenic properties and morphology traits in this nutrient-intense crop.

Baby salad leaves of salad roquette, arugula in the U.S. (Eruca vesicaria ssp. sativa) had an increased postharvest shelf life of 2 to 6 days, while lollo rosso lettuce (Lactuca sativa L. `Ravita') and red chard [Beta vulgaris L. var. flavescens (Lam.) Lam] baby salad leaves had increased shelf life of 1 to 2 days when harvested at the end of the day compared with leaves harvested at the start of the day. We have shown that improved shelf life of salad roquette and lollo rosso following end-of-day harvest was correlated with altered biophysical characteristics of the cell walls, with increased cell wall extensibility (percent plasticity and elasticity) measured at end of day. Leaf turgor pressure (P, MPa) was also highest in salad roquette and red chard at the end of day. Improved shelf life following `end of day' harvest was also associated with the accumulation of leaf sucrose in salad roquette but not lollo rosso and red chard following daily photosynthesis. Diurnal alterations of leaf starch concentration were detected in lollo rosso and red chard but not in salad roquette. The degree of leaf shelf life extension in salad roquette and red chard was further associated with the peak rates of leaf photosynthetic activity. These data suggest that, depending on species, significant improvements to postharvest shelf life could be achieved through the rescheduling of time of day for harvest and also provide relevant information on the selection of traits for future genetic improvement.

Using a free-air CO2 enrichment (FACE) experiment, poplar trees (Populus · euramericana clone I214) were exposed to either ambient or elevated (CO2) from planting, for a 5-year period during canopy development, closure, coppice and re-growth. In each year, measurements were taken of stomatal density (SD, number mm � 2 ) and stomatal index (SI, the proportion of epidermal cells forming stomata). In year 5, measurements were also taken of leaf stomatal conductance (gs, lmol m � 2 s � 1 ), photosynthetic CO2 fixation (A, mmol m � 2 s � 1 ), instantaneous water-use efficiency (A/E) and the ratio of intercellular to atmospheric CO2 (Ci:Ca). Elevated (CO2) caused reductions in SI in the first year, and in SD in the first 2 years, when the canopy was largely open. In fol- lowing years, when the canopy had closed, elevated (CO2) had no detectable effects on stomatal numbers or index. In contrast, even after 5 years of exposure to elevated (CO2), gs was reduced, A/E was stimulated, and Ci:Ca was reduced relative to ambient (CO2). These outcomes from the long-term realistic field conditions of this forest FACE experiment suggest that stomatal numbers (SD and SI) had no role in determining the improved instantaneous leaf-level efficiency of water use under elevated (CO2). We pro- pose that altered cuticular development during canopy closure may partially explain the changing response of stomata to elevated (CO2), although the mechanism for this remains obscure.

The ‘processability’ of baby salad leaves may be defined as the ability to withstand the postharvest washing and packing processes that are involved in the production of ready-to-eat bagged salads. The inability of baby salad leaves (species including Lactuca sativa L. and Spinacia oleracea L.) to withstand processing results in a reduction in crop shelf-life. Leaves from geographically diverse locations displayed strikingly different processability scores from visual inspection. We have shown that these ‘good’ and ‘poor’ quality leaves may be differentiated from assessments of the biophysical properties of the cell wall (% plasticity) and epidermal cell size. Artificial manipulation of processability in the glasshouse through the application of a mechanical stress or a high salt stress produced L. sativa cv. Ravita (a leaf type ‘lollo rosso’ lettuce) leaves covering a range of processability. Mechanical stress, applied as a daily dose of 100 paper strokes, increased lettuce leaf shelf-life by 33% and was associated with reduced % plasticity and smaller leaf epidermal cells. These traits are thus proposed to be of key importance in the description of processability, with the plant cell wall and plant cell wall gene expression implicated. The potential for future manipulation of these traits for the pre-packed salad market is considered.

Thermal imaging has been used in the past for remote detection of regions of canopy showing symptoms of stress, including water deficit stress. Stress indices derived from thermal images have been used as an indicator of canopy water status, but these depend on the choice of reference surfaces and environmental conditions and can be confounded by variations in complex canopy structure. Therefore, in this work, instead of using stress indices, information from thermal and visible light imagery was combined along with machine learning techniques to identify regions of canopy showing a response to soil water deficit. Thermal and visible light images of a spinach canopy with different levels of soil moisture were captured. Statistical measurements from these images were extracted and used to classify between canopies growing in well-watered soil or under soil moisture deficit using Support Vector Machines (SVM) and Gaussian Processes Classifier (GPC) and a combination of both the classifiers. The classification results show a high correlation with soil moisture. We demonstrate that regions of a spinach crop responding to soil water deficit can be identified by using machine learning techniques with a high accuracy of 97%. This method could, in principle, be applied to any crop at a range of scales. Horticultural Development Company (HDC) and by the Department of Computer Science, University of Warwick. Vitacress Salads Ltd. Biotechnology and Biological Sciences Research Council (BBSRC). Scopus