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QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure–activity relationships and understanding the mechanisms by which these compounds promote the human immune response. Vaccine immunoadjuvants are central to vaccine efficiency. Now, the complete characterization of the biosynthetic pathway of QS-21, a potent immunoadjuvant produced by the Chilean soapbark tree, has been reported. These findings open the door to heterologous production of QS-21 and new-to-nature adjuvants.

The past 40 years in Southeast Asia have seen about 50% of lowland rainforests converted to oil palm and other plantations, and much of the remaining forest heavily logged. Little is known about how fragmentation influences recovery and whether climate change will hamper restoration. Here, we use repeat airborne LiDAR surveys spanning the hot and dry 2015-16 El Niño Southern Oscillation event to measure canopy height growth across 3,300 ha of regenerating tropical forests spanning a logging intensity gradient in Malaysian Borneo. We show that the drought led to increased leaf shedding and branch fall. Short forest, regenerating after heavy logging, continued to grow despite higher evaporative demand, except when it was located close to oil palm plantations. Edge effects from the plantations extended over 300 metres into the forests. Forest growth on hilltops and slopes was particularly impacted by the combination of fragmentation and drought, but even riparian forests located within 40 m of oil palm plantations lost canopy height during the drought. Our results suggest that small patches of logged forest within plantation landscapes will be slow to recover, particularly as ENSO events are becoming more frequent. It is unclear whether tropical forest fragments within plantation landscapes are resilient to drought. Here the authors analyse LiDAR and ground-based data from the 2015-16 El Niño event across a logging intensity gradient in Borneo. Although regenerating forests continued to grow, canopy height near oil palm plantations decreased, and a strong edge effect extended up to at least 300 m away.

Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop’s association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce β-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin.

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family—as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.

Old‐growth forests in Southeast Asia are dominated by trees of the Dipterocarpaceae family which are targeted by selective logging. Their traits (supra‐annual mast fruiting, limited dispersal, and recalcitrant seeds that form no seed bank) mean they can have poor natural regeneration rates in some selectively logged forests. Enrichment planting is used to attempt to overcome this recruitment limitation and increase restoration success. However, it is still unclear what factors influence the performance of planted seedlings. Here, we analyse the growth and survival between 2012 and 2015 of 721 enrichment line‐planted seedlings from 16 species of dipterocarps within the selectively logged forest of the Sabah Biodiversity Experiment, alongside the location, size and identity of nearly 5000 naturally occurring trees within 10 m of focal planted seedlings. We analysed the survival and growth of enrichment planted dipterocarp seedlings in relation to three properties of the surrounding naturally occurring vegetation: (1) canopy openness; (2) the abundance of naturally occurring dipterocarps (proportion of total basal area); (3) the presence of nearby (< 10 m) large trees (basal area of the largest tree as a proportion of the total). Survival and growth rates of enrichment planted seedlings were positively associated with canopy openness and total basal area of surrounding trees. These results were consistent between the two planting cohorts. Increased survival and growth of enrichment planted seedlings in areas of Sabah Biodiversity Experiment with higher canopy openness (up to around 40%) is consistent with understory light as a limiting resource. The unexpected higher survival and growth of enrichment planted seedlings in forest areas with higher basal area of unlogged trees may be explained by the creation of patches that are better or worse for tree growth, with areas less heavily impacted by logging containing both more naturally occurring trees and providing better conditions for the survival and growth of planted seedlings. This study analyzed the growth and survival of 721 seedlings in Malaysian Borneo in relation to their local neighbourhood of surrounding matrix trees. We found that both were positively influenced by canopy openness and the basal area of surrounding trees, highlighting the importance of light availability and the benefits of areas with more unlogged trees in supporting seedling development.

Background Grass pea ( Lathyrus sativus ) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. Results A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for β-L-ODAP-quantification. This method utilises an internal standard (di- 13 C-labelled β-L-ODAP) allowing accurate quantification of β-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any β-L-ODAP in these species. The LCMS method was also used to quantify β-L-ODAP accurately in different tissues of grass pea. Conclusions The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and β-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of β-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new ‘gold standard’ for β-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-β-L-ODAP genotypes.

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-Hyp 4 , serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Ara f s), 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 Ara f to Hyp-arabinofuranotriose (Hyp-β1,4Ara f -β1,2Ara f -β1,2Ara f ) side chains in an α-linkage, to yield Hyp-Ara f 4 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-Ara f 4 , point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.