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Salicylic acid (SA) and pipecolic acid (Pip) play important roles in plant immunity. Here we analyzed the roles of transcription factors TGACG-BINDING FACTOR 1 (TGA1) and TGA4 in regulating SA and Pip biosynthesis in Arabidopsis thaliana. We quantified the expression levels of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g), which encode two master transcription factors of plant immunity, and the accumulation of SA and Pip in tga1-1 tga4-1 mutant plants. We tested whether SARD1 and CBP60g are direct targets of TGA1 by chromatin immunoprecipitation–polymerase chain reaction (ChIP-PCR). In addition to promoting pathogen-induced SA biosynthesis, we found that SARD1 and CBP60g also positively regulated Pip biosynthesis by targeting genes encoding key biosynthesis enzymes of Pip. TGA1/TGA4 were required for full induction of SARD1 and CBP60g in plant defense. ChIP-PCR analysis showed that SARD1 was a direct target of TGA1. In tga1-1 tga4-1 mutant plants, the expression levels of SARD1 and CBP60g along with SA and Pip accumulation following pathogen infection were dramatically reduced compared with those in wild-type plants. Consistent with reduced expression of SARD1 and CBP60g, pathogen-associated molecular pattern (PAMP)-induced pathogen resistance and systemic acquired resistance were compromised in tga1-1 tga4-1. Our study showed that TGA1 and TGA4 regulate Pip and SA biosynthesis by modulating the expression of SARD1 and CBP60g.

Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 (SARD4), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana. Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ1-piperideine-2-carboxylic acid (P2C). In Escherichia coli, expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use L-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment.

Premise Recently, plant science has seen transformative advances in scalable data collection for sequence and chemical data. These large datasets, combined with machine learning, have demonstrated that conducting plant metabolic research on large scales yields remarkable insights. A key next step in increasing scale has been revealed with the advent of accessible large language models, which, even in their early stages, can distill structured data from the literature. This brings us closer to creating specialized databases that consolidate virtually all published knowledge on a topic. Methods Here, we first test different combinations of prompt engineering techniques and language models in the identification of validated enzyme–product pairs. Next, we evaluate the application of automated prompt engineering and retrieval‐augmented generation to identify compound–species associations. Finally, we build and determine the accuracy of a multimodal language model–based pipeline that transcribes images of tables into machine‐readable formats. Results When tuned for each specific task, these methods perform with high (80–90%) or modest (50%) accuracies for enzyme–product pair identification and table image transcription, but with lower false‐negative rates than previous methods (decreasing from 55% to 40%) for compound–species pair identification. Discussion We enumerate several suggestions for researchers working with language models, among which is the importance of the user's domain‐specific expertise and knowledge.

Cork is a water-impermeable, suberin-based material harboring lignin, (hemi)cellulose, and extractable small molecules (primarily triterpenoids). Extractables strongly influence the properties of suberin-based materials. Though these previous findings suggest a key role for triterpenoids in cork material quality, directly testing this idea is hindered in part because it is not known which genes control cork triterpenoid biosynthesis. Here, we used gas chromatography and mass spectrometry to determine that the majority (>85%) of non-polar extractables from cork were pentacyclic triterpenoids, primarily betulinic acid, friedelin, and hydroxy-friedelin. In other plants, triterpenoids are generated by oxidosqualene cyclases (OSCs). Accordingly, we mined Quercus suber EST libraries for OSC fragments to use in a RACE PCR-based approach and cloned three full-length OSC transcripts from cork ( QsOSC1 - 3 ). Heterologous expression in Saccharomyces cerevisiae revealed that QsOSC1 - 3 respectively encoded enzymes with lupeol synthase, mixed α- and β-amyrin synthase, and mixed β-amyrin and friedelin synthase activities. These activities together account for the backbone structures of the major cork triterpenoids. Finally, we analyzed the sequences of QsOSC1-3 and other plant OSCs to identify residues associated with specific OSC activities, then combined this with analyses of Q. suber transcriptomic and genomic data to evaluate potential redundancies in cork triterpenoid biosynthesis.

Transformer-based large language models are receiving considerable attention because of their ability to analyse scientific literature. Small language models (SLMs), however, also have potential in this area as they have smaller compute footprints and allow users to keep data in-house. Here, we quantitatively evaluate the ability of SLMs to: (i) score references according to project-specific relevance and (ii) extract and structuring data from unstructured sources (scientific abstracts). By comparing SLMs’ outputs against those of a human on hundreds of abstracts, we found that (i) SLMs can effectively filter literature and extract structured information relatively accurately (error rates as low as 10%), but not with perfect yield (as low as 50% in some cases), (ii) that there are tradeoffs between accuracy, model size and computing requirements and (iii) that clearly written abstracts are needed to support accurate data extraction. We recommend advanced prompt engineering techniques, full-text resources and model distillation as future directions.

Premise Specialized metabolites serve various roles for plants and humans. Unlike core metabolites, specialized metabolites are restricted to certain plant lineages; thus, in addition to their ecological functions, specialized metabolites can serve as diagnostic markers of plant lineages. Methods We investigated the phylogenetic distribution of plant metabolites using non‐proteogenic amino acids (NPAA). Species–NPAA associations for eight NPAAs were identified from the existing literature and placed within a phylogenetic context using R packages and the Interactive Tree of Life. To confirm and extend the literature‐based NPAA distribution, we selected azetidine‐2‐carboxylic acid (Aze) and screened over 70 diverse plants using gas chromatography–mass spectrometry (GC‐MS). Results Literature searches identified 163 NPAA‐relevant articles, which were manually inspected to identify 822 species–NPAA associations. NPAAs were mapped at the order and genus level, revealing that some NPAAs are restricted to single orders, whereas others are present across divergent taxa. The observed distribution of Aze across plants and ancestral state reconstruction suggests a convergent evolutionary history. Discussion Although reliance on chemotaxonomy has decreased in recent years, there is still value in placing metabolites within a phylogenetic context to understand the evolutionary processes of plant chemical diversification. This approach can be applied to metabolites present in any organism and compared at a range of taxonomic levels.

Plants use chemistry to overcome diverse challenges. A particularly striking chemical trait that some plants possess is the ability to synthesize massive amounts of epicuticular wax that accumulates on the plant's surfaces as a white coating visible to the naked eye. The ability to synthesize basic wax molecules appears to be shared among virtually all land plants, and our knowledge of ubiquitous wax compound synthesis is reasonably advanced. However, the ability to synthesize thick layers of visible epicuticular crystals (“wax blooms”) is restricted to specific lineages, and our knowledge of how wax blooms differ from ubiquitous wax layers is less developed. Here, we recruited the help of citizen scientists and middle school students to survey the wax bloom chemistry of 78 species spanning dicot, monocot, and gymnosperm lineages. Using gas chromatography–mass spectrometry, we found that the major wax classes reported from bulk wax mixtures can be present in wax bloom crystals, with fatty acids, fatty alcohols, and alkanes being present in many species' bloom crystals. In contrast, other compounds including aldehydes, ketones, secondary alcohols, and triterpenoids were present in only a few species' wax bloom crystals. By mapping the 78 wax bloom chemical profiles onto a phylogeny and using phylogenetic comparative analyses, we found that secondary alcohol and triterpenoid‐rich wax blooms were present in lineage‐specific patterns that would not be expected to arise by chance. That finding is consistent with reports that secondary alcohol biosynthesis enzymes are found only in certain lineages but was a surprise for triterpenoids, which are intracellular components in virtually all plant lineages. Thus, our data suggest that a lineage‐specific mechanism other than biosynthesis exists that enables select species to generate triterpenoid‐rich surface wax crystals. Overall, our study outlines a general mode in which research scientists can collaborate with citizen scientists as well as middle and high school classrooms not only to enhance data collection and generate testable hypotheses but also to directly involve classrooms in the scientific process and inspire future STEM workers.

Cuticular waxes coat aerial plant surfaces to protect tissues against biotic and abiotic stress. The waxes are complex mixtures of fatty-acid-derived lipids formed on modular biosynthetic pathways, with varying chain lengths and oxygen functional groups. The waxes of most plant species contain C 26 –C 32 alcohols, aldehydes, alkanes, and fatty acids together with their alkyl esters, and comparisons between diverse wax mixtures have revealed matching chain length distributions between some of these compound classes. Based on such patterns, the biosynthetic pathways leading to the ubiquitous wax constituents were hypothesized early on, and most of these pathway hypotheses have since been confirmed by biochemical and molecular genetic studies in model species. However, the most abundant wax compounds on many species, including many important crop species, contain secondary functional groups and thus their biosynthesis differs at least in part from the ubiquitous wax compounds with which they co-occur. Here, we survey the chemical structures of these species-specific specialty wax compounds based on a comprehensive CAS SciFinder search and then review relevant reports on wax compositions to help develop and refine hypotheses for their biosynthesis. Across the plant kingdom, specialty wax compounds with one, two, and three secondary functional groups have been identified, with most studies focusing on Angiosperms. Where multiple specialty wax compounds were reported, they frequently occurred as homologous series and/or mixtures of isomers. Among these, it is now possible to recognize series of homologs with predominantly odd- or even-numbered chain lengths, and mixtures of isomers with functional groups on adjacent or on alternating carbon atoms. Using these characteristic molecular geometries of the co-occurring specialty compounds, they can be categorized and, based on the common structural patterns, mechanisms of biosynthesis may be predicted. It seems highly likely that mixtures of isomers with secondary functions on adjacent carbons arise from oxidation catalyzed by P450 enzymes, while mixtures of isomers with alternating group positions are formed by malonate condensation reactions mediated by polyketide synthase or ketoacyl-CoA synthase enzymes, or else by the head-to-head condensation of long-chain acyls. Though it is possible that some enzymes leading to ubiquitous compounds also participate in specialty wax compound biosynthesis, comparisons between co-occurring ubiquitous and specialty wax compounds strongly suggest that, at least in some species, dedicated specialty wax compound machinery exists. This seems particularly true for the diverse species in which specialty wax compounds, most notably nonacosan-10-ol, hentriacontan-16-one (palmitone), and very-long-chain β-diketones, accumulate to high concentrations.

Epidermal cells bear a wax-sealed cuticle to hinder transpirational water loss. The amount and composition of the cuticular wax mixture may change as organs develop, to optimize the cuticle for specific functions during growth. Here, morphometrics, wax chemical profiling, and gene expression measurements were integrated to study developing Arabidopsis thaliana leaves and, thus, further our understanding of cuticular wax ontogeny. Before 5 days of age, cells at the leaf tip ceased dividing and began to expand, while cells at the leaf base switched from cycling to expansion at day 13, generating a cell age gradient along the leaf. We used this spatial age distribution together with leaves of different ages to determine that, as leaves developed, their wax compositions shifted from C₂₄/C₂₆ to C₃₀/C₃₂ and from fatty acid to alkane constituents. These compositional changes paralleled an increase in the expression of the elongase enzyme CER6 but not of alkane pathway enzymes, suggesting that CER6 transcriptional regulation is responsible for both chemical shifts. Leaves bore constant numbers of trichomes between 5 and 21 days of age and, thus, trichome density was higher on young leaves. During this time span, leaves of the trichome-less gl1 mutant had constant wax coverage, while wild-type leaf coverage was initially high and then decreased, suggesting that high trichome density leads to greater apparent coverage on young leaves. Conversely, wax coverage on pavement cells remained constant over time, indicating that wax accumulation is synchronized with cell expansion throughout leaf development.

Main conclusionQuantification of cuticular waxes coupled with insect bioassays and feeding behavior analysis demonstrate that long-chain C32 fatty alcohol impacts host plant selection by aphids.Cuticular waxes constitute the first point of contact between plants and their environment, and it also protect plants from external stresses. However, the role of waxes in Sorghum bicolor (sorghum) against sugarcane aphid (Melanaphis sacchari), a relatively new and devastating pest of sorghum in the U.S., is not fully understood. In this study, we monitored sugarcane aphid behavior on two genotypes of young sorghum plants with different wax chemistry: a wild-type plant (bloom) with lower C32 alcohol cuticular wax, and a mutant plant (bloomless) with 1.6 times the amount of wax compared to wild-type plants. No-choice aphid bioassays revealed that sugarcane aphid reproduction did not vary between wild-type and the bloomless plants. Electrical Penetration Graph (EPG) monitoring indicated that the sugarcane aphids spent comparable amount of time feeding from the sieve elements of the wild-type and bloomless plants. However, aphids spent more time feeding on the xylem sap of the bloomless plants compared to the wild-type plants. Furthermore, aphid choice assays revealed that the sugarcane aphids preferred to settle on bloomless compared to wild-type plants. Overall, our results suggest that cuticular waxes on young sorghum leaves play a critical role in influencing host plant selection by sugarcane aphids.