De novo genome assembly of white clover (Trifolium repens L.) reveals the role of copy number variation in rapid environmental adaptation.

BACKGROUND: White clover (Trifolium repens) is a globally important perennial forage legume. This species also serves as an eco-evolutionary model system for studying within-species chemical defense variation; it features a well-studied polymorphism for cyanogenesis (HCN release following tissue damage), with higher frequencies of cyanogenic plants favored in warmer locations worldwide. Using a newly generated haplotype-resolved genome and two other long-read assemblies, we tested the hypothesis that copy number variants (CNVs) at cyanogenesis genes play a role in the ability of white clover to rapidly adapt to local environments. We also examined questions on subgenome evolution in this recently evolved allotetraploid species and on chromosomal rearrangements in the broader IRLC legume clade. RESULTS: Integration of PacBio HiFi, Omni-C, Illumina, and linkage map data yielded a completely de novo genome assembly for white clover (created without a priori sequence assignment to subgenomes). We find that white clover has undergone extensive transposon diversification since its origin but otherwise shows highly conserved genome organization and composition with its diploid progenitors. Unlike some other clover species, its chromosomal structure is conserved with other IRLC legumes. We further find extensive evidence of CNVs at the major cyanogenesis loci; these contribute to quantitative variation in the cyanogenic phenotype and to local adaptation across wild North American populations. CONCLUSIONS: This work provides a case study documenting the role of CNVs in local adaptation in a plant species, and it highlights the value of pan-genome data for identifying contributions of structural variants to adaptation in nature.

Beyond cyanogenesis: Temperature gradients drive environmental adaptation in North American white clover (Trifolium repens L.).

Species that repeatedly evolve phenotypic clines across environmental gradients have been highlighted as ideal systems for characterizing the genomic basis of local environmental adaptation. However, few studies have assessed the importance of observed phenotypic clines for local adaptation: conspicuous traits that vary clinally may not necessarily be the most critical in determining local fitness. The present study was designed to fill this gap, using a plant species characterized by repeatedly evolved adaptive phenotypic clines. White clover is naturally polymorphic for its chemical defence cyanogenesis (HCN release with tissue damage); climate-associated cyanogenesis clines have evolved throughout its native and introduced range worldwide. We performed landscape genomic analyses on 415 wild genotypes from 43 locations spanning much of the North American species range to assess the relative importance of cyanogenesis loci vs. other genomic factors in local climatic adaptation. We find clear evidence of local adaptation, with temperature-related climatic variables best describing genome-wide differentiation between sampling locations. The same climatic variables are also strongly correlated with cyanogenesis frequencies and gene copy number variations (CNVs) at cyanogenesis loci. However, landscape genomic analyses indicate no significant contribution of cyanogenesis loci to local adaptation. Instead, several genomic regions containing promising candidate genes for plant response to seasonal cues are identified - some of which are shared with previously identified QTLs for locally adaptive fitness traits in North American white clover. Our findings suggest that local adaptation in white clover is likely determined primarily by genes controlling the timing of growth and flowering in response to local seasonal cues. More generally, this work suggests that caution is warranted when considering the importance of conspicuous phenotypic clines as primary determinants of local adaptation.

Exploration and utilization of novel aldoxime, nitrile, and nitro compounds metabolizing enzymes from plants and arthropods.

Aldoxime (R1R2C=NOH) and nitrile (R-C≡N) are nitrogen-containing compounds that are found in species representing all kingdoms of life. The enzymes discovered from the microbial "aldoxime-nitrile" pathway (aldoxime dehydratase, nitrile hydratase, amidase, and nitrilase) have been thoroughly studied because of their industrial importance. Although plants utilize cytochrome P450 monooxygenases to produce aldoxime and nitrile, many biosynthetic pathways are yet to be studied. Cyanogenic millipedes accumulate various nitrile compounds, such as mandelonitrile. However, no such aldoxime- and nitrile-metabolizing enzymes have been identified in millipedes. Here, I review the exploration of novel enzymes from plants and millipedes with characteristics distinct from those of microbial enzymes, the catalysis of industrially useful reactions, and applications of these enzymes for nitrile compound production.

Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens).

PREMISE: ß-Cyanoalanine synthase (ß-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of ß-CAS and Aox genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species. METHODS: We used a natural polymorphism for cyanogenesis in white clover to examine ß-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure. We identified all ß-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a, and Aox2d subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue. RESULTS: ß-CAS and Aox2a expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression. Aox2d, in contrast, appeared to be upregulated as a generalized wounding response. CONCLUSIONS: These findings suggest a heightened constitutive role for HCN detoxification (via elevated ß-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a expression) in plants that are capable of cyanogenesis. As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.

Cyanogenesis, a Plant Defence Strategy against Herbivores.

Plants and phytophagous arthropods have coevolved in a long battle for survival. Plants respond to phytophagous feeders by producing a battery of antiherbivore chemical defences, while herbivores try to adapt to their hosts by attenuating the toxic effect of the defence compounds. Cyanogenic glucosides are a widespread group of defence chemicals that come from cyanogenic plants. Among the non-cyanogenic ones, the Brassicaceae family has evolved an alternative cyanogenic pathway to produce cyanohydrin as a way to expand defences. When a plant tissue is disrupted by an herbivore attack, cyanogenic substrates are brought into contact with degrading enzymes that cause the release of toxic hydrogen cyanide and derived carbonyl compounds. In this review, we focus our attention on the plant metabolic pathways linked to cyanogenesis to generate cyanide. It also highlights the role of cyanogenesis as a key defence mechanism of plants to fight against herbivore arthropods, and we discuss the potential of cyanogenesis-derived molecules as alternative strategies for pest control.

Transcript profiles of wild and domesticated sorghum under water-stressed conditions and the differential impact on dhurrin metabolism.

MAIN CONCLUSION: Australian native species of sorghum contain negligible amounts of dhurrin in their leaves and the cyanogenesis process is regulated differently under water-stress in comparison to domesticated sorghum species. Cyanogenesis in forage sorghum is a major concern in agriculture as the leaves of domesticated sorghum are potentially toxic to livestock, especially at times of drought which induces increased production of the cyanogenic glucoside dhurrin. The wild sorghum species endemic to Australia have a negligible content of dhurrin in the above ground tissues and thus represent a potential resource for key agricultural traits like low toxicity. In this study we investigated the differential expression of cyanogenesis related genes in the leaf tissue of the domesticated species Sorghum bicolor and the Australian native wild species Sorghum macrospermum grown in glasshouse-controlled water-stress conditions using RNA-Seq analysis to analyse gene expression. The study identified genes, including those in the cyanogenesis pathway, that were differentially regulated in response to water-stress in domesticated and wild sorghum. In the domesticated sorghum, dhurrin content was significantly higher compared to that in the wild sorghum and increased with stress and decreased with age whereas in wild sorghum the dhurrin content remained negligible. The key genes in dhurrin biosynthesis, CYP79A1, CYP71E1 and UGT85B1, were shown to be highly expressed in S. bicolor. DHR and HNL encoding the dhurrinase and α-hydroxynitrilase catalysing bio-activation of dhurrin were also highly expressed in S. bicolor. Analysis of the differences in expression of cyanogenesis related genes between domesticated and wild sorghum species may allow the use of these genetic resources to produce more acyanogenic varieties in the future.

Genetic trade-offs underlie divergent life history strategies for local adaptation in white clover.

Local adaptation is common in plants, yet characterization of its underlying genetic basis is rare in herbaceous perennials. Moreover, while many plant species exhibit intraspecific chemical defence polymorphisms, their importance for local adaptation remains poorly understood. We examined the genetic architecture of local adaptation in a perennial, obligately-outcrossing herbaceous legume, white clover (Trifolium repens). This widespread species displays a well-studied chemical defence polymorphism for cyanogenesis (HCN release following tissue damage) and has evolved climate-associated cyanogenesis clines throughout its range. Two biparental F2  mapping populations, derived from three parents collected in environments spanning the U.S. latitudinal species range (Duluth, MN, St. Louis, MO and Gainesville, FL), were grown in triplicate for two years in reciprocal common garden experiments in the parental environments (6,012 total plants). Vegetative growth and reproductive fitness traits displayed trade-offs across reciprocal environments, indicating local adaptation. Genetic mapping of fitness traits revealed a genetic architecture characterized by allelic trade-offs between environments, with 100% and 80% of fitness QTL in the two mapping populations showing significant QTL×E interactions, consistent with antagonistic pleiotropy. Across the genome there were three hotspots of QTL colocalization. Unexpectedly, we found little evidence that the cyanogenesis polymorphism contributes to local adaptation. Instead, divergent life history strategies in reciprocal environments were major fitness determinants: selection favoured early investment in flowering at the cost of multiyear survival in the southernmost site versus delayed flowering and multiyear persistence in the northern environments. Our findings demonstrate that multilocus genetic trade-offs contribute to contrasting life history characteristics that allow for local adaptation in this outcrossing herbaceous perennial.

Unique and highly specific cyanogenic glycoside localization in stigmatic cells and pollen in the genus Lomatia (Proteaceae).

BACKGROUND AND AIMS: Floral chemical defence strategies remain understudied despite the significance of flowers to plant fitness, and the fact that many flowers contain secondary metabolites that confer resistance to herbivores. Optimal defence and apparency theories predict that the most apparent plant parts and/or those most important to fitness should be most defended. To test whether within-flower distributions of chemical defence are consistent with these theories we used cyanogenic glycosides (CNglycs), which are constitutive defence metabolites that deter herbivores by releasing hydrogen cyanide upon hydrolysis. METHODS: We used cyanogenic florets of the genus Lomatia to investigate at what scale there may be strategic allocation of CNglycs in flowers, what their localization reveals about function, and whether levels of floral CNglycs differ between eight congeneric species across a climatic gradient. Within-flower distributions of CNglycs during development were quantified, CNglycs were identified and their localization was visualized in cryosectioned florets using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). KEY RESULTS: Florets of all congeneric species studied were cyanogenic, and concentrations differed between species. Within florets there was substantial variation in CNglyc concentrations, with extremely high concentrations (up to 14.6 mg CN g-1 d. wt) in pollen and loose, specialized surface cells on the pollen presenter, among the highest concentrations reported in plant tissues. Two tyrosine-derived CNglycs, the monoglycoside dhurrin and diglycoside proteacin, were identified. MALDI-MSI revealed their varying ratios in different floral tissues; proteacin was primarily localized to anthers and ovules, and dhurrin to specialized cells on the pollen presenter. The mix of transient specialized cells and pollen of L. fraxinifolia was ~11 % dhurrin and ~1.1 % proteacin by mass. CONCLUSIONS: Tissue-specific distributions of two CNglycs and substantial variation in their concentrations within florets suggests their allocation is under strong selection. Localized, high CNglyc concentrations in transient cells challenge the predictions of defence theories, and highlight the importance of fine-scale metabolite visualization, and the need for further investigation into the ecological and metabolic roles of CNglycs in floral tissues.

Storage and release of hydrogen cyanide in a chelicerate (Oribatula tibialis).

Cyanogenesis denotes a chemical defensive strategy where hydrogen cyanide (HCN, hydrocyanic or prussic acid) is produced, stored, and released toward an attacking enemy. The high toxicity and volatility of HCN requires both chemical stabilization for storage and prevention of accidental self-poisoning. The few known cyanogenic animals are exclusively mandibulate arthropods (certain myriapods and insects) that store HCN as cyanogenic glycosides, lipids, or cyanohydrins. Here, we show that cyanogenesis has also evolved in the speciose Chelicerata. The oribatid mite Oribatula tibialis uses the cyanogenic aromatic ester mandelonitrile hexanoate (MNH) for HCN storage, which degrades via two different pathways, both of which release HCN. MNH is emitted from exocrine opisthonotal oil glands, which are potent organs for chemical defense in most oribatid mites.

Role of cyanogenic glycosides in the seeds of wild lima bean, Phaseolus lunatus: defense, plant nutrition or both?

MAIN CONCLUSION: Cyanogenic glycosides present in the seeds of wild lima bean plants are associated with seedling defense but do not affect seed germination and seedling growth. Wild lima bean plants contain cyanogenic glycosides (CNGs) that are known to defend the plant against leaf herbivores. However, seed feeders appear to be unaffected despite the high levels of CNGs in the seeds. We investigated a possible role of CNGs in seeds as nitrogen storage compounds that influence plant growth, as well as seedling resistance to herbivores. Using seeds from four different wild lima bean natural populations that are known to vary in CNG levels, we tested two non-mutually exclusive hypotheses: (1) seeds with higher levels of CNGs produce seedlings that are more resistant against generalist herbivores and, (2) seeds with higher levels of CNGs germinate faster and produce plants that exhibit better growth. Levels of CNGs in the seeds were negatively correlated with germination rates and not correlated with seedling growth. However, levels of CNGs increased significantly soon after germination and seeds with the highest CNG levels produced seedlings with higher CNG levels in cotyledons. Moreover, the growth rate of the generalist herbivore Spodoptera littoralis was lower in cotyledons with high-CNG levels. We conclude that CNGs in lima bean seeds do not play a role in seed germination and seedling growth, but are associated with seedling defense. Our results provide insight into the potential dual function of plant secondary metabolites as defense compounds and storage molecules for growth and development.

Herbivores and plant defences affect selection on plant reproductive traits more strongly than pollinators.

Pollinators and herbivores can both affect the evolutionary diversification of plant reproductive traits. However, plant defences frequently alter antagonistic and mutualistic interactions, and therefore, variation in plant defences may alter patterns of herbivore- and pollinator-mediated selection on plant traits. We tested this hypothesis by conducting a common garden field experiment using 50 clonal genotypes of white clover (Trifolium repens) that varied in a Mendelian-inherited chemical antiherbivore defence-the production of hydrogen cyanide (HCN). To evaluate whether plant defences alter herbivore- and/or pollinator-mediated selection, we factorially crossed chemical defence (25 cyanogenic and 25 acyanogenic genotypes), herbivore damage (herbivore suppression) and pollination (hand pollination). We found that herbivores weakened selection for increased inflorescence production, suggesting that large displays are costly in the presence of herbivores. In addition, herbivores weakened selection on flower size but only among acyanogenic plants, suggesting that plant defences reduce the strength of herbivore-mediated selection. Pollinators did not independently affect selection on any trait, although pollinators weakened selection for later flowering among cyanogenic plants. Overall, cyanogenic plant defences consistently increased the strength of positive directional selection on reproductive traits. Herbivores and pollinators both strengthened and weakened the strength of selection on reproductive traits, although herbivores imposed ~2.7× stronger selection than pollinators across all traits. Contrary to the view that pollinators are the most important agents of selection on reproductive traits, our data show that selection on reproductive traits is driven primarily by variation in herbivory and plant defences in this system.

Metabolomics and Biochemical Approaches Link Salicylic Acid Biosynthesis to Cyanogenesis in Peach Plants.

Despite the long-established importance of salicylic acid (SA) in plant stress responses and other biological processes, its biosynthetic pathways have not been fully characterized. The proposed synthesis of SA originates from chorismate by two distinct pathways: the isochorismate and phenylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. To date, however, no relationship has been suggested between the two pathways. In this work, by metabolomics and biochemical approaches (including the use of [13C]-labeled compounds), we provide strong evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions. The main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from phenylalanine, controlling their turnover. In peach plants MD is the intermediary molecule of the suggested new SA biosynthetic pathway and CNglcs turnover, regulating the biosynthesis of both amygdalin and SA. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway). MD also provided partial protection against Plum pox virus infection in peach seedlings. Thus, we propose a third pathway, an alternative to the PAL pathway, for SA synthesis in peach plants.

Continent-Wide Climatic Variation Drives Local Adaptation in North American White Clover.

Climate-associated clines in adaptive polymorphisms are commonly cited as evidence of local adaptation within species. However, the contribution of the clinally varying trait to overall fitness is often unknown. To address this question, we examined survival, vegetative growth, and reproductive output in a central US common garden experiment using 161 genotypes of white clover (Trifolium repens L.) originating from 15 locations across North America. White clover is polymorphic for cyanogenesis (hydrogen cyanide release upon tissue damage), a chemical defense against generalist herbivores, and climate-associated cyanogenesis clines have repeatedly evolved across the species range. Over a 12-month experiment, we observed striking correlations between the population of origin and plant performance in the common garden, with climatic distance from the common garden site predicting fitness more accurately than geographic distance. Assessments of herbivore leaf damage over the 2015 growing season indicated marginally lower herbivory on cyanogenic plants; however, this effect did not result in increased fitness in the common garden location. Linear mixed modeling suggested that while cyanogenesis variation had little predictive value for vegetative growth, it is as important as climatic variation for predicting reproductive output in the central United States. Together, our findings suggest that knowledge of climate similarity, as well as knowledge of locally favored adaptive traits, will help to inform transplantation strategies for restoration ecology and other conservation efforts in the face of climate change.

Urbanization drives the evolution of parallel clines in plant populations.

Urban ecosystems are an increasingly dominant feature of terrestrial landscapes. While evidence that species can adapt to urban environments is accumulating, the mechanisms through which urbanization imposes natural selection on populations are poorly understood. The identification of adaptive phenotypic changes (i.e. clines) along urbanization gradients would facilitate our understanding of the selective factors driving adaptation in cities. Here, we test for phenotypic clines in urban ecosystems by sampling the frequency of a Mendelian-inherited trait-cyanogenesis-in white clover (Trifolium repens L.) populations along urbanization gradients in four cities. Cyanogenesis protects plants from herbivores, but reduces tolerance to freezing temperatures. We found that the frequency of cyanogenic plants within populations decreased towards the urban centre in three of four cities. A field experiment indicated that spatial variation in herbivory is unlikely to explain these clines. Rather, colder minimum winter ground temperatures in urban areas compared with non-urban areas, caused by reduced snow cover in cities, may select against cyanogenesis. In the city with no cline, high snow cover might protect plants from freezing damage in the city centre. Our study suggests that populations are adapting to urbanization gradients, but regional climatic patterns may ultimately determine whether adaptation occurs.

Interactive effects of temperature and drought on cassava growth and toxicity: implications for food security?

Cassava is an important dietary component for over 1 billion people, and its ability to yield under drought has led to it being promoted as an important crop for food security under climate change. Despite its known photosynthetic plasticity in response to temperature, little is known about how temperature affects plant toxicity or about interactions between temperature and drought, which is important because cassava tissues contain high levels of toxic cyanogenic glucosides, a major health and food safety concern. In a controlled glasshouse experiment, plants were grown at 2 daytime temperatures (23 °C and 34 °C), and either well-watered or subject to a 1 month drought prior to harvest at 6 months. The objective was to determine the separate and interactive effects of temperature and drought on growth and toxicity. Both temperature and drought affected cassava physiology and chemistry. While temperature alone drove differences in plant height and above-ground biomass, drought and temperature × drought interactions most affected tuber yield, as well as foliar and tuber chemistry, including C : N, nitrogen and cyanide potential (CNp; total cyanide released from cyanogenic glucosides). Conditions that most stimulated growth and yield (well-watered × high temperature) effected a reduction in tuber toxicity, whereas drought inhibited growth and yield, and was associated with increased foliar and tuber toxicity. The magnitude of drought effects on tuber yield and toxicity were greater at high temperature; thus, increases in tuber CNp were not merely a consequence of reduced tuber biomass. Findings confirm that cassava is adaptable to forecast temperature increases, particularly in areas of adequate or increasing rainfall; however, in regions forecast for increased incidence of drought, the effects of drought on both food quality (tuber toxicity) and yield are a greater threat to future food security and indicate an increasing necessity for processing of cassava to reduce toxicity.

The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous ß-glucosidases resulting from a crucial amino acid substitution.

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α-hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific ß-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic γ- and ß-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only ß-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic ß-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.

Lotus japonicus flowers are defended by a cyanogenic ß-glucosidase with highly restricted expression to essential reproductive organs.

Flowers and leaves of Lotus japonicus contain α-, ß-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by ß-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

Utilization of a high-throughput shoot imaging system to examine the dynamic phenotypic responses of a C4 cereal crop plant to nitrogen and water deficiency over time.

The use of high-throughput phenotyping systems and non-destructive imaging is widely regarded as a key technology allowing scientists and breeders to develop crops with the ability to perform well under diverse environmental conditions. However, many of these phenotyping studies have been optimized using the model plant Arabidopsis thaliana. In this study, The Plant Accelerator(®) at The University of Adelaide, Australia, was used to investigate the growth and phenotypic response of the important cereal crop, Sorghum bicolor L. Moench and related hybrids to water-limited conditions and different levels of fertilizer. Imaging in different spectral ranges was used to monitor plant composition, chlorophyll, and moisture content. Phenotypic image analysis accurately measured plant biomass. The data set obtained enabled the responses of the different sorghum varieties to the experimental treatments to be differentiated and modelled. Plant architectural instead of architecture elements were determined using imaging and found to correlate with an improved tolerance to stress, for example diurnal leaf curling and leaf area index. Analysis of colour images revealed that leaf 'greenness' correlated with foliar nitrogen and chlorophyll, while near infrared reflectance (NIR) analysis was a good predictor of water content and leaf thickness, and correlated with plant moisture content. It is shown that imaging sorghum using a high-throughput system can accurately identify and differentiate between growth and specific phenotypic traits. R scripts for robust, parsimonious models are provided to allow other users of phenomic imaging systems to extract useful data readily, and thus relieve a bottleneck in phenotypic screening of multiple genotypes of key crop plants.

Age versus stage: does ontogeny modify the effect of phosphorus and arbuscular mycorrhizas on above- and below-ground defence in forage sorghum?

Arbuscular mycorrhizas (AM) can increase plant acquisition of P and N. No published studies have investigated the impact of P and AM on the allocation of N to the plant defence, cyanogenic glucosides. We investigated the effects of soil P and AM on cyanogenic glucoside (dhurrin) concentration in roots and shoots of two forage sorghum lines differing in cyanogenic potential (HCNp). Two harvest times allowed plants grown at high and low P to be compared at the same age and the same size, to take account of known ontogenetic changes in shoot HCNp. P responses were dependent on ontogeny and tissue type. At the same age, P-limited plants were smaller and had higher shoot HCNp but lower root HCNp. Ontogenetically controlled comparisons showed a P effect of lesser magnitude, and that there was also an increase in the allocation of N to dhurrin in shoots of P-limited plants. Colonization by AM had little effect on shoot HCNp, but increased root HCNp and the allocation of N to dhurrin in roots. Divergent responses of roots and shoots to P, AM and with ontogeny demonstrate the importance of broadening the predominantly foliar focus of plant defence studies/theory, and of ontogenetically controlled comparisons.

Adaptive cyanogenesis clines evolve recurrently through geographical sorting of existing gene deletions.

Identifying the genetic basis of parallel phenotypic evolution provides insight into the process of adaptation and evolutionary constraint. White clover (Trifolium repens) has evolved climate-associated adaptive clines in cyanogenesis (the ability to produce hydrogen cyanide upon tissue damage) in several world regions where it has been introduced. Gene-deletion polymorphisms at the CYP79D15 and Li loci underlie the presence/absence of the cyanogenic phenotype. Both loci have undergone multiple independent gene-deletion events, which are identifiable through molecular signatures in flanking regions. To investigate whether cyanogenesis clines in introduced populations have evolved through the sorting of standing genetic variation or de novo gene deletions, we examined cyanogenesis gene-flanking regions in three world regions. In comparison with native Eurasian populations, we find no evidence for novel gene deletion events in any introduced region, which suggests that these adaptive clines have evolved through the geographical sorting of pre-existing genetic variation.