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bioRxiv2020

A molecular toolkit for the green seaweed Ulva mutabilis

The green seaweed Ulva is an ecologically-important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed Ulva mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed. One-sentence summaryMolecular cloning tools allow to generate gain-of-function seaweed lines that will help to study seaweed biology.

plant biology↗

bioRxiv2019

Comparative analysis of chemically and biologically synthesized iron oxide nanoparticles against Leishmania tropica

The current study was carried out to compare the antileishmanial potential of the characterized chemically and plant mediated (Trigonella foenum-graecum) FeO-NPs (FeO-NPs) against L.tropica KWH23. The promastigotes and amastigotes mortality, ROS generation, and biocompatibility assessment were estimated in time-dose dependent manner by PDT. LED exposed bio-inspired FeO-NPs after 72 hours, express significant suppressive effects by exhibiting 50% inhibitory concentration (IC50) 0.001572{+/-}0.02g/ml and 0.011408{+/-}0.02g/ml for promastigotes and amastigotes, respectively. Leishmanial DNA damage and membrane integrity caused by FeO-NPs were owe to high production of reactive oxygen species (ROS). By applying different ROS scavengers (mannitol and sodium azide) hydroxyl radical and singlet oxygen were found as main moieties for cell death by giving quantum yield 0.15 and 0.28 through chemically and green synthesized FeO-NPs, respectively. The light exposed green synthesized nanoparticles were found biocompatible on human red blood cells (RBCs), by exhibiting LD50 value 2659g/mL. From these findings, it can be concluded that plant mediated FeO-NPs can be used as promising antiprotozoal agent.

plant biology↗

bioRxiv2025

The minor antennae of photosystem II contribute to qH-energy dissipation in Arabidopsis

Photosynthesis is a biological process that converts light energy into chemical energy. Excessive light can damage the photosynthetic machinery, so plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). Among the NPQ mechanisms, qH is a form of sustained quenching, dependent on LIPOCALIN IN THE PLASTID (LCNP) and repressed by SUPPRESSOR OF QUENCHING 1 (SOQ1), protecting against abiotic stress. Recently, we showed in Arabidopsis thaliana that qH can occur in the major light-harvesting complexes (Lhcb1, Lhcb2, Lhcb3) but independently of any specific major antenna. Interestingly, in mutants with little or no accumulation of major antennae (koLHCII, lhcb1, cpsrp43), qH can still be induced. Here, we show that the minor antennae can be quenched by qH and remain quenched once isolated. To investigate the role of minor antennae in qH, we combined the soq1 mutant, which displays high qH, with mutations in each minor antenna type (Lhcb4, Lhcb5, or Lhcb6), or with a mutant lacking all minor antennae. None are strictly required for qH to occur. Still, the absence of Lhcb6 decreases qH induction likely due to an indirect effect from the slower electron transport rate and/or a different macro-organization of photosynthetic complexes in the thylakoids. Overall, this work demonstrates that the minor antennae are a secondary target for qH and could serve as an additional safety valve for photoprotective energy dissipation during prolonged stress.

plant biology↗

bioRxiv2023

A novel phenylpropanoid methyl esterase enables catabolism of aromatic compounds that inhibit biological nitrification

Agriculture is the largest source of greenhouse gases (GHG) production. Conversion of nitrogen fertilizers into more reduced forms by microbes through a process known as biological nitrification drives GHG production, enhances proliferation of toxic algal blooms, and increases cost of crop production. Some plants reduce biological nitrification in soils by exuding a diverse array of biological nitrification inhibitors (BNIs) that inhibit the ammonium oxidizing microbes responsible for nitrification. Applying synthetic biology to enhance and transfer BNI production into food and bioenergy crops is a promising approach to reduce nitrification but the success of this strategy is contingent upon improving our limited understanding of BNIs mechanisms-of-action and degradation in the soil. We addressed this gap by investigating the previously unknown metabolic route through which a prominent class of aromatic BNIs known as phenylpropanoid methyl esters (PPMEs) are catabolized. While neither transcriptomics (RNAseq) or high-throughput functional genomics (RB-TnSeq) methods reduced the genetic search space for pathway discovery into a tenable number of genes, the combination of narrowed the search space to a collection of 4 proteins of unknown function. Using genetic and biochemical analyses we found that two previously uncharacterized enzymes, including a novel phenylpropanoid methyl esterase, funnel PPMEs into an established phenylpropanoid catabolism pathway. Transfer of these two enzymes into bacteria capable of using other phenylpropanoids use of PPMEs as carbon sources. This work both provided insight into BNI catabolism and is the first step towards development of model in vivo plant-microbe systems for studying BNI mechanisms under well controlled conditions.

microbiology↗

bioRxiv2020

Temporal dynamics of QTL effects on vegetative growth in Arabidopsis thaliana

We assessed early vegetative growth in a population of 382 accessions of Arabidopsis thaliana using automated non-invasive high-throughput phenotyping. All accessions were imaged daily from seven to 18 days after sowing in three independent experiments and genotyped using the Affymetrix 250k SNP array. Projected leaf area (PLA) was derived from image analysis and used to calculate relative growth rates (RGR). In addition, initial seed size was determined. The generated data sets were used jointly for a genome-wide association study that identified 238 marker-trait associations (MTAs) individually explaining up to 8 % of the total phenotypic variation. Co-localisation of MTAs occurred at 33 genomic positions. At 21 of these positions, sequential co-localisation of MTAs for two to nine consecutive days was observed. The detected MTAs for PLA and RGR could be grouped according to their temporal expression patterns, emphasising that temporal variation of MTA action can be observed even during the vegetative growth phase, a period of continuous formation and enlargement of seemingly similar rosette leaves. This indicates that causal genes may be differentially expressed in successive periods. Analyses of the temporal dynamics of biological processes are needed to gain important insight into the molecular mechanisms of growth-controlling processes in plants. HighlightA genome-wide association study including the factor time highlighted that early plant growth in Arabidopsis is governed by several medium and many small effect loci, most of which act only during short phases of two to nine days.

plant biology↗

bioRxiv2025

Pea-Saving Partners: Bacillus and Pseudomonas combat downy mildew in pea crops

Downy mildew (DM) is a destructive disease that significantly reduces the yield and quality of important pulses (legumes) and horticultural crops, particularly during humid and cool seasons. This disease is caused by obligate and host-specific oomycete pathogens. Controlling the pathogen is challenging due to its long-term survival as spores and its rapid mutation. Use of chemical pesticides has been the most effective method to control DM pathogens, but their environmental hazards are a global concern. Current research is focused on exploring the potential of microbial biological control agents (MBCA), particularly rhizobacteria strains of the genera Bacillus and Pseudomonas, which have shown suppression of plant pathogens. However, to date, no MBCA has been reported to be effective against DM pathogens in pulses. We investigated the effectiveness of Bacillus and Pseudomonas strains as potential biopesticides against the pea downy mildew pathogen Peronospora viciae f. sp. pisi (Pvp). In our study, in vitro bioassays showed 100% inhibition of Pvp spore germination compared to the control. In planta antagonism assays further demonstrated significant suppression (>80%) of Pvp sporulation in pea plants sprayed with strains of Bacillus velezensis or P. fluorescens or their filtrates. The drench application also showed significant effects where either a Pseudomonas or cold-adapted Bacillus strain was used. We observed a synergistic effect for the dual foliar application of the microbes compared to individual application (27.6 to 46.7% suppression). Furthermore, the results from the molecular biomass analysis were consistent with the results of the sporulation assays. This demonstrates the strong interactive and promotive benefits of using Bacillus and Pseudomonas as biocontrol agents Based on these results, we conclude that these MBCAs could be effective in combatting Pvp infections in the field.

plant biology↗

bioRxiv2019

Composition variation of agarwood-associated microbial communities from Aquilaria sinensis

Agarwood, derived from Aquilaria sinensis and Aquilaria malaccensis, is of medicinal and ecological value and religious importance as incense. The existing imbalance between short supply and increasing demand of this product remains to be solved. Thus, the biologically artificial agarwood-inducing methods commonly called whole-tree agarwood-inducing techniques (agar-wit) have been established to dramatically improve agarwood yield within a short period. However, several studies reported a lower content of ethanol-soluble extractive in the agar-wit agarwood than in the natural agarwood. To further understand the role of microorganisms in agarwood formation, we investigated and contrasted the endophytic bacteria and fungi between different types of agarwood from A. sinensis through high-throughput sequencing. Results showed that the same dominant phyla of bacteria consisting of Proteobacteria, Actinobacteria, and Acidobacteria were shared by the natural agarwood and agar-wit agarwood. Meanwhile, Ascomycota and Basidiomycota constituted the similar dominant fungal phyla of these two kinds of agarwood. However, the principal microbial communities at the genus or order level evidently varied from natural agarwood to agar-wit agarwood. Moreover, the bacterial communities are closely connected with terpenoid and carbohydrate metabolism, which indicated that the bacterial communities also play a vital role in agarwood formation. In conclusion, the higher concentrated abundance of the dominant microbial communities in agar-wit agarwood than in natural agarwood may promote agarwood formation, however, the low evenness of microbial communities also lowers the content of ethanol-soluble extractive.\n\nIMPORTANCEAgarwood has become an indispensable product in modern life because of medicinal value, ecological and religious importance as incense. Nevertheless, the enormous demand for agarwood markedly exceeds the supply because of the dramatically declining population of genus Aquilaria. Agarwood formation occurring slowly and infrequently in a natural environment, so various artificial techniques were developed to promote the formation of agarwood, such as the physical methods and chemical methods. However, these techniques still are insufficient to compensate for the agarwood shortage. In this case, a novel biological method called the whole-tree agarwood-inducing technique (Agar-wit) induces agarwood production. However, several studies have shown that agarwood harvested from biological technology contains lower content of ethanol-soluble extractive compared with natural agarwood. So to further expose and understand the endophytic bacteria and fungi in agarwood formation is important for the improvement of biological method.

plant biology↗

bioRxiv2019

Inference of Gene Regulatory Network Uncovers the Linkage Between Circadian Clock and Crassulacean Acid Metabolism in Kalanchoë fedtschenkoi

The circadian clock drives time-specific gene expression, allowing for associated biological processes to be active during certain times of the 24 h day. Crassulacean acid metabolism (CAM) photosynthetic plants represent an interesting case of circadian regulation of gene expression as CO2 fixation and stomatal movement in CAM plants display strong circadian dynamics. The molecular mechanisms behind how the circadian clock enabled these physiological differences is not well understood. Therefore, we set out to investigate whether core circadian elements in CAM plants were re-phased during evolution, or whether networks of phase-specific genes were simply connected to different core elements. We utilized a new metric for identifying candidate core genes of a periodic gene network and then applied the Local Edge Machine (LEM) algorithm to infer regulatory relationships between the candidate core clock genes and orthologs of known core clock genes in K. fedtschenkoi. We also used LEM to identify stomata-related gene targets for K. fedtschenkoi core clock genes and constructed a subsequent gene regulatory network. Our results provide new insights into the mechanism of circadian control of CAM-related genes in K. fedtschenkoi, facilitating the engineering of CAM machinery into non-CAM plants for sustainable crop production in water-limited environments.

plant biology↗

bioRxiv2020

RNA editing in inflorescences of wild grapevine unveils association to sex and development

RNA editing challenges the central dogma of molecular biology, by modifying the genetic information at the transcription level. Recent reports, suggesting increased levels of RNA editing in plants, raised questions on the nature and dynamics of such events during development. We here report the occurrence of distinct RNA editing patterns in wild Vitis flowers during development, with twelve possible RNA editing modifications observed for the first time in plants. RNA editing events are gender and developmental stage specific, identical in subsequent years of this perennial species and with distinct nucleotide frequencies neighboring editing sites on the 5 and 3 flanks. The transcriptome dynamics unveils a new regulatory layer responsible for gender plasticity enhancement or underling dioecy evolution in Vitis.

plant biology↗

bioRxiv2021

A simple Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the {beta}-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

plant biology↗

bioRxiv2026

Host Plant and Light Regime Shape Limited, Whole-Body Transcriptional Rhythmicity in the Bird Cherry-Oat Aphid (Rhopalosiphum padi)

Background Daily cycles in environmental conditions shape the biology of plants and their insect herbivores. Because aphids feed nearly continuously from the phloem to sustain rapid population growth, they maintain prolonged, direct contact with their hosts, which exposes them to plant-derived daily rhythms in addition to those arising from the environment. We investigated whole-body transcriptional rhythmicity in the bird cherry-oat aphid (Rhopalosiphum padi) under light-dark (LD) and constant darkness (DD) conditions while aphids fed on either wheat or an artificial diet sachet, sampling transcript expression every 4 h over a 48 h window. Results Only a small fraction of the transcriptome in R. padi displayed 24 h rhythms, and this fraction declined further as light and host cues were removed. Unexpectedly, more transcripts were rhythmic in DD than in LD on wheat. This trend was not present in diet-fed aphids and was not consistently detected across rhythmic detection tools or significance thresholds. Transcripts that were rhythmic in aphids feeding on wheat hosts had higher amplitudes compared to those feeding on diet. Peak expression (i.e., phase) for rhythmic transcripts clustered near dawn and pre-dusk (zeitgeber time, ZT0 and ZT12), except for transcripts detected as rhythmic in darkness-only on wheat, which peaked approximately 4 h later. Rhythmic transcripts detected in this study were functionally enriched for processes relevant to plant-aphid interactions, including carbohydrate metabolism and detoxification. Subsequent promoter analysis found E-box motifs in both rhythmic and non-rhythmic transcripts, and de novo promoter motif discovery on rhythmic transcripts identified recurring Mad/SMAD- and PAR-bZIP-like motifs. Conclusions Whole-body transcriptional rhythmicity in R. padi is limited in scope and appears strongly dependent on environmental and host-derived cues. The candidate transcripts and regulatory pathways identified in this study provide clear targets for tissue-specific, functionally minded follow-up studies to investigate the extent and regulatory mechanisms of endogenous physiological and behavioral rhythms in R. padi.

molecular biology↗

bioRxiv2020

Integration of Transcriptome and Small RNA Sequencing to Decipher Molecular Interaction of Chenopodium quinoa Varieties with Cucumber Mosaic Virus

Saponins are secondary metabolites with antiviral properties. Low saponin (sweet) varieties of quinoa (Chenopodium quinoa) have been developed because seeds high in saponins taste bitter. The aim of this study was to elucidate the role of saponin in resistance of quinoa to Cucumber mosaic virus (CMV). Differential gene expression was studied in time-series study of CMV infection. High-throughput transcriptome sequence data were obtained from 36 samples (3 varieties x +/- CMV x 1 or 4 days after inoculation x 3 replicates). Translation, lipid, nitrogen, amino acid metabolism, and mono- and sesquiterpenoid biosynthesis genes were upregulated in CMV infections. In Red Head, (bitter), CMV-induced systemic symptoms were concurrent with downregulation of a key saponin biosynthesis gene, TSARL1, four days after inoculation. In local lesion responses (sweet and semi-sweet), TSARL1 levels remained up-regulated. Known microRNAs (miRNA) (81) from 11 miR families and 876 predicted novel miRNAs were identified. Differentially expressed miRNA and short interfering RNA clusters (24nt) induced by CMV infection are predicted to target genomic and intergenic regions enriched in repetitive elements. This is the first report of integrated RNASeq and sRNASeq data in quinoa-virus interactions and provides comprehensive understanding of involved genes, non-coding regions, and biological pathways in virus resistance.

plant biology↗

bioRxiv2023

Trading acyls and swapping sugars: metabolic innovations in Solanum trichomes

Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced by AcylSugar AcylTransferases from sugars and acyl-coenzyme A esters. Published research revealed trichome acylsugars composed of glucose and sucrose cores in species across the family. In addition, acylsugars were analyzed across a small fraction of the >1200 species in the phenotypically megadiverse Solanum genus, with a handful containing inositol and glycosylated inositol cores. The current study sampled several dozen species across subclades of the Solanum to get a more detailed view of acylsugar chemodiversity. In depth characterization of acylsugars from the Clade II species Solanum melongena (brinjal eggplant) led to the identification of eight unusual structures with inositol or inositol glycoside cores, and hydroxyacyl chains. Liquid chromatography-mass spectrometry analysis of 31 additional species in the Solanum genus revealed striking acylsugar diversity with some traits restricted to specific clades and species. Acylinositols and inositol-based acyldisaccharides were detected throughout much of the genus. In contrast, acylglucoses and acylsucroses were more restricted in distribution. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to the identification of the S. melongena AcylSugar AcylTransferase 3-Like 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and appears to be a functionally divergent ASAT3. This study provides a foundation for investigating the evolution and function of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology.

plant biology↗

bioRxiv2025

Utilization of Arabidopsis E3 ubiquitin decoys high-throughput yeast screen platform to dissect the ubiquitin-mediated circadian clock regulation

Protein ubiquitination, mediated by E3 ubiquitin ligases, is a critical regulatory mechanism of eukaryotic cellular processes, including circadian clock function. However, identifying E3-substrate pairs remains technically challenging due to substrate instability and the genetic redundancy of E3s. To overcome these limitations, we developed a high-throughput yeast two-hybrid E3 decoy screening platform, enabling systematic mapping of E3-substrate interactions. Using a library of 283 Arabidopsis F-box and U-box E3 decoys, we screened 21 core circadian clock regulators and identified 77 potential E3-substrate interaction pairs involving 56 E3s and 16 clock proteins. Focusing on high-confidence hits, we demonstrated that PUB18 physically interacts with the central clock regulators LHY and JMJD5 and promotes their ubiquitination in planta. Genetic analyses further revealed that PUB18 and its homolog PUB19 function redundantly in circadian clock regulation. This study establishes the E3 decoy yeast two-hybrid platform as a versatile and scalable tool for dissecting ubiquitination networks in broad biological processes.

plant biology↗

bioRxiv2021

Institutional Differences in the Stewardship and Research Output of United States Herbaria

Public policy decisions regarding institutional frameworks that govern the stewardship of biodiversity data at public and private institutions are an area of increasing importance. Museums, government agencies, and academic institutions across the United States maintain collections of biological specimens and information critical to scientific discovery. One subset of these natural history collections are herbaria, or collections of preserved plant matter and their associated data. In this study, I evaluate the current state of the digitization and databasing of herbariums contributing data to the SEInet Regional Network of North American Herbaria, and assess the impact of characteristics, particularly institution type (cultural sector institutions, public universities, private universities, or public land institutions), on the metrics of herbaria richness, digitization, and research usage. The results of this study suggest that institution type is significantly associated with the size, diversity, and digitization efforts of a herbarium collection. Specifically, cultural sector institutions tend to have larger and more diverse collections, followed by public and private universities, and finally public land institutions. Additionally, as herbarium size and richness increases, the research output of associated staff also increases. These results highlight that some institutions, particularly larger institutions located at universities or cultural sector institutions, may be better supported in the curation, stewardship, and digitization of large collections, allowing long-term access to the associated biodiversity data. Smaller institutions at public land institutions may need additional support in these endeavors, and may represent an area of unmet needs for digitization and curatorial funding and resources.

plant biology↗

bioRxiv2025

Biological nitrification inhibition (BNI) in wheat for climate adaptation in acidic and alkaline soils

Anthropogenic disturbances to nitrogen (N) cycling, particularly through agricultural N use, have intensified nitrous oxide (N2O) emissions. Developing wheat lines with biological nitrification inhibition (BNI) is a promising strategy to reduce such emissions. However, the effectiveness of BNI depends not only on plant characteristics, but also on how they interact with environmental factors such as soil pH and elevated atmospheric CO2 (eCO2). This study evaluated the response of a BNI-capable wheat line (Triticum aestivum cv. ROELFS) to eCO2 under contrasting soil pH during the 30 days following ammonium fertilization. Two wheat lines (ROELFS-Control and ROELFS-BNI) were grown under ambient (400 ppm) and elevated (800 ppm) CO2 in acidic (pH 5.3) and alkaline (pH 8.8) soils. N2O emissions, nitrifying and denitrifying microbial communities, and soil chemical properties were monitored to assess plant-soil-microbe interactions. ROELFS-BNI consistently reduced N2O emissions under all conditions by reducing archaeal (Nitrososphaeraceae) and bacterial nitrifiers (Nitrosomonadaceae, Nitrospiraceae) without major shifts in overall microbial composition, indicating high specificity of BNI exudates. Elevated CO2 effects on N2O emissions were pH-dependent. In acidic soil, eCO2 increased emissions in ROELFS-Control but not in ROELFS-BNI, likely due to the enrichment of complete denitrifiers (e.g. Rhodanobacteraceae). However, in alkaline soil, eCO2 reduced N2O emissions in both Control and BNI lines, especially the latter, which was associated with a higher abundance of N2O-reducing denitrifiers (e.g. Burkholderiaceae). This study highlights the potential of ROELFS-BNI wheat as a sustainable practice to mitigate N pollution adaptable to diverse soils and predicted CO2 atmospheric conditions.

plant biology↗

bioRxiv2025

A photoprotection dial maps holistic light-stress response in diatoms

Cellular responses to light stress operate on two timescales: short-term responses, driven by dynamic photosynthetic mechanisms such as non-photochemical quenching (NPQ), and long-term responses, characterized by various physiological and metabolic shifts driven by changes in gene expression. Understanding the coordination within and between these layers represents a major challenge in photosynthesis research. We achieved this in the diatom Phaeodactylum tricornutum using an adjustable "photoprotection dial" across 10 mutant strains expressing different constitutive levels of the Lhcx1 protein, which determine their latent NPQ capacity. Crucially, a near-identical initial state is preserved among strains, which allows a precise mapping of the causal chain of events initiated by NPQ induction under high light. This unlocks exceptional leverage to examine holistic interactions between functional photosynthesis and gene expression within their natural regulatory architecture. We observe that increasing NPQ maintains the PSII primary acceptor QA in a more oxidized state with little effect on linear electron flow. This oxidation limits PSII photodamage and promotes cyclic electron flow around PSI, which in turn is consistent with enhanced ATP production supporting PSII repair. Moreover, transcriptomic analysis across high-light exposure time suggests that the expression of nearly half of the genome is modulated via NPQ-driven effects. Gene expression profile clustering reveals distinct and coherent transcriptomic regulatory networks. The short-term response is characterized by a strong downregulation of light-harvesting-related genes -- independently of NPQ -- and an upregulation of oxidative stress response genes that scales inversely with NPQ magnitude. In the long term, most of the transcriptome is deeply impacted by high-light stress in a widely NPQ-dependent manner, highlighting the critical role of this process in shaping photophysiology, which in turn influences gene expression. By engineering a native functional feedback loop into an experimental dial, our approach establishes a generalizable framework for studying the complex interplay between physiology and system-wide biological regulation.

plant biology↗

bioRxiv2026

Comparison of Extraction Methods for the Quantification of Phytohormones from Tomato Fruits and Leaves by LC-MS/MS

Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the complex system of phytohormone signaling pathways. Quantification of phytohormones with the commonly used technique liquid chromatography coupled to tandem mass spectrometry is susceptible to the influence of non-phytohormone components present in the sample, a phenomenon referred to as matrix effect. To reduce matrix effect, some phytohormone quantification methods include additional steps of cleanup of crude extracts. However, to what extent additional purification steps provide increased accuracy compared to simpler, less laborious methods is seldomly evaluated. We evaluated three previously described phytohormone extraction methods, two of which include solid-phase extraction and one that does not, in their ability to minimize matrix effect and generate accurate estimates of phytohormone species spanning six classifications, from fruit and leaf tissue of Solanum lycopersicum cv. Micro-Tom (tomato). Our results show that, while the methods that included solid phase extraction occasionally outperformed each other regarding matrix effect and/or recovery efficiency for broad range of phytohormones, they rarely outperformed the simpler single-phase extraction method. Short AbstractAccurate, simultaneous quantification of chemically diverse phytohormones by LC-MS/MS is frequently confounded by matrix effects, leading to the incorporation of additional purification steps. We systematically compared three published extraction protocols with or without solid-phase extraction in tomato tissues across six hormone classes. Solid-phase methods occasionally improved matrix suppression or recovery, but did not consistently outperform the single-phase approach, questioning the added value of extra cleanup steps, particularly when high-throughput is desired, as in the case of systems biology interrogations.

plant biology↗