Enhanced stress resilience in potato by deletion of Parakletos.

Continued climate change impose multiple stressors on crops, including pathogens, salt, and drought, severely impacting agricultural productivity. Innovative solutions are necessary to develop resilient crops. Here, using quantitative potato proteomics, we identify Parakletos, a thylakoid protein that contributes to disease susceptibility. We show that knockout or silencing of Parakletos enhances resistance to oomycete, fungi, bacteria, salt, and drought, whereas its overexpression reduces resistance. In response to biotic stimuli, Parakletos-overexpressing plants exhibit reduced amplitude of reactive oxygen species and Ca2+ signalling, and silencing Parakletos does the opposite. Parakletos homologues have been identified in all major crops. Consecutive years of field trials demonstrate that Parakletos deletion enhances resistance to Phytophthora infestans and increases yield. These findings demark a susceptibility gene, which can be exploited to enhance crop resilience towards abiotic and biotic stresses in a low-input agriculture.

Arabinosylation of cell wall extensin is required for the directional response to salinity in roots.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.

Transcriptomics and starch biosynthesis analysis in leaves and developing seeds of mung bean provide a basis for genetic engineering of starch composition and seed quality.

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

Cytosine base editors optimized for genome editing in potato protoplasts.

In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3-4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.

Effects of growth temperature on multi-scale structure of root tuber starch in sweet potato.

Sweet potato was planted at three soil and air temperatures (21, 25 and 28 °C) with the same humidity and light time/intensity. Root tuber starches were isolated, and their multi-scale structures were investigated to reveal the effects of growth temperature on starch properties. Growth temperature did not change the morphology and amylose content of starch, but markedly increased the size of starch from volume-weighted mean diameter 12.2 µm to 17.0 µm. Starch grown at high growth temperature exhibited less A branch-chains and lower branching degree of amylopectin and more B2 and B3+ branch-chains of amylopectin than at low growth temperature. With increasing growth temperature, starch changed from CC-type to CA-type, its relative crystallinity and lamellar peak intensity increased, and the thickness of crystalline and amorphous lamellae did not significantly change. Starch grown at high growth temperature exhibited significantly higher gelatinization temperature than at low growth temperature, but had similar gelatinization enthalpy.

Corrigendum: Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).

[This corrects the article DOI: 10.3389/fgeed.2021.795644.].

Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes for In Vitro Shoot Regeneration From Tissues and Protoplasts of Potato and Rapeseed: Implications of Bioengineering Such as Gene Editing of Broad-Leaved Plants.

Schemes for efficient regenerationand recovery of shoots from in vitro tissues or single cells, such as protoplasts, are only available for limited numbers of plant species and genotypes and are crucial for establishing gene editing tools on a broader scale in agriculture and plant biology. Growth conditions, including hormone and nutrient composition as well as light regimes in key steps of known regeneration protocols, display significant variations, even between the genotypes within the same species, e.g., potato (Solanum tuberosum). As fresh plant material is a prerequisite for successful shoot regeneration, the plant material often needs to be refreshed for optimizing the growth and physiological state prior to genetic transformation. Utilization of protoplasts has become a more important approach for obtaining transgene-free edited plants by genome editing, CRISPR/Cas9. In this approach, callus formation from protoplasts is induced by one set of hormones, followed by organogenesis, i.e., shoot formation, which is induced by a second set of hormones. The requirements on culture conditions at these key steps vary considerably between the species and genotypes, which often require quantitative adjustments of medium compositions. In this mini-review, we outline the protocols and notes for clonal regeneration and cultivation from single cells, particularly protoplasts in potato and rapeseed. We focus mainly on different hormone treatment schemes and highlight the importance of medium compositions, e.g., sugar, nutrient, and light regimes as well as culture durations at the key regeneration steps. We believe that this review would provide important information and hints for establishing efficient regeneration strategies from other closely related and broad-leaved plant species in general.

Plant Protein O-Arabinosylation.

A wide range of proteins with diverse functions in development, defense, and stress responses are O-arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O-glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O-glycosylation of the Hyp C4-OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (ArafTs) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all ArafT enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O-glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O-arabinosylation. Here, we provide an update on the prevalence and types of O-arabinosylated proteins and the enzymatic machinery responsible for their modifications.

Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes.

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.

Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).

Potato, Solanum tuberosum is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the gene encoding glucan water dikinase (GWD) 1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3-2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. At the same time, high editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids. Furthermore, high editing efficiencies reduce the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNPs) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and the DMR6-1, 6-10 gRNAs were designed to target regions comprising the 5' and the 3' end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs varied significantly, and some generated specific indel patterns. RNP's targeting the 5' end of GWD1 yielded significantly higher editing efficiency as compared to targeting the 3' end. For DMR6-1, such an effect was not seen. Simultaneously targeting each of the two target regions with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3' end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or a slightly negative effect on editing from the single or combined gRNA/RNPs. These initial findings may instigate much larger studies needed for facilitating and optimizing precision breeding in plants.

INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

Engineering mammalian cells to produce plant-specific N-glycosylation on proteins.

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and ß2xylose (ß2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/ß2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

Phenolic cross-links: building and de-constructing the plant cell wall.

Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2nd generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.

High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato.

CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.

Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts.

BACKGROUND: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. RESULTS: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. CONCLUSIONS: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.

Extensin arabinoside chain length is modulated in elongating cotton fibre.

Cotton fibre provides a unicellular model system for studying cell expansion and secondary cell wall deposition. Mature cotton fibres are mainly composed of cellulose while the walls of developing fibre cells contain a variety of polysaccharides and proteoglycans required for cell expansion. This includes hydroxyproline-rich glycoproteins (HRGPs) comprising the subgroup, extensins. In this study, extensin occurrence in cotton fibres was assessed using carbohydrate immunomicroarrays, mass spectrometry and monosaccharide profiling. Extensin amounts in three species appeared to correlate with fibre quality. Fibre cell expression profiling of the four cotton cultivars, combined with extensin arabinoside chain length measurements during fibre development, demonstrated that arabinoside side-chain length is modulated during development. Implications and mechanisms of extensin side-chain length dynamics during development are discussed.

Spatial separation of the cyanogenic ß-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of Zygaena larvae facilitates cyanide release.

Low molecular weight compounds are typically used by insects and plants for defence against predators. They are often stored as inactive ß-glucosides and kept separate from activating ß-glucosidases. When the two components are mixed, the ß-glucosides are hydrolysed releasing toxic aglucones. Cyanogenic plants contain cyanogenic glucosides and release hydrogen cyanide due to such a well-characterized two-component system. Some arthropods are also cyanogenic, but comparatively little is known about their system. Here, we identify a specific ß-glucosidase (ZfBGD2) involved in cyanogenesis from larvae of Zygaena filipendulae (Lepidoptera, Zygaenidae), and analyse the spatial organization of cyanide release in this specialized insect. High levels of ZfBGD2 mRNA and protein were found in haemocytes by transcriptomic and proteomic profiling. Heterologous expression in insect cells showed that ZfBGD2 hydrolyses linamarin and lotaustralin, the two cyanogenic glucosides present in Z. filipendulae. Linamarin and lotaustralin as well as cyanide release were found exclusively in the haemoplasma. Phylogenetic analyses revealed that ZfBGD2 clusters with other insect ß-glucosidases, and correspondingly, the ability to hydrolyse cyanogenic glucosides catalysed by a specific ß-glucosidase evolved convergently in insects and plants. The spatial separation of the ß-glucosidase ZfBGD2 and its cyanogenic substrates within the haemolymph provides the basis for cyanide release in Z. filipendulae. This spatial separation is similar to the compartmentalization of the two components found in cyanogenic plant species, and illustrates one similarity in cyanide-based defence in these two kingdoms of life.

Corrigendum: Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

This corrects the article DOI: 10.1038/srep45341.

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD.

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1-5 ß- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-ß1,4Araf-ß1,2Araf-ß1,2Araf) side chains in an α-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its' product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.

Lepidopteran defence droplets - a composite physical and chemical weapon against potential predators.

Insects often release noxious substances for their defence. Larvae of Zygaena filipendulae (Lepidoptera) secrete viscous and cyanogenic glucoside-containing droplets, whose effectiveness was associated with their physical and chemical properties. The droplets glued mandibles and legs of potential predators together and immobilised them. Droplets were characterised by a matrix of an aqueous solution of glycine-rich peptides (H-WG11-NH2) with significant amounts of proteins and glucose. Among the proteins, defensive proteins such as protease inhibitors, proteases and oxidases were abundant. The neurotoxin ß-cyanoalanine was also found in the droplets. Despite the presence of cyanogenic glucosides, which release toxic hydrogen cyanide after hydrolysis by a specific ß-glucosidase, the only ß-glucosidase identified in the droplets (ZfBGD1) was inactive against cyanogenic glucosides. Accordingly, droplets did not release hydrogen cyanide, unless they were mixed with specific ß-glucosidases present in the Zygaena haemolymph. Droplets secreted onto the cuticle hardened and formed sharp crystalline-like precipitates that may act as mandible abrasives to chewing predators. Hardening followed water evaporation and formation of antiparallel ß-sheets of the peptide oligomers. Consequently, after mild irritation, Zygaena larvae deter predators by viscous and hardening droplets that contain defence proteins and ß-cyanoalanine. After severe injury, droplets may mix with exuding haemolymph to release hydrogen cyanide.