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bioRxiv2024

An analysis of natural variation in Pinus pinaster through the lens of systems biology

Pinus pinaster is a main species in Mediterranean forests, being naturally distributed through the Mediterranean basin, covering regions with a variety of geoclimatic conditions. This distribution in different environments leads, by natural selection, to a natural variation within the species that manifests at phenotypic level in populations with different growth features and overall tree architectures. Studying P. pinaster natural variation is necessary to understand the genetic heritage of the species and can provide valuable information for information-based decision-making regarding forest management and breeding programmes. In this paper, we analyzed the natural variation in needles and buds from three provenances from contrasting geoclimatic locations using a common garden approach and proteomics. The integration of the proteomics data with tree growth-related parameters, geoclimatic features at provenances original locations, and sample-matched metabolomics data previously generated provided novel knowledge on metabolism rearrangements related to secondary metabolism and associated to growth features and the adaptation to light and UV-B radiation intensities.

plant biology↗

bioRxiv2020

UGT76B1, a promiscuous hub of small molecule-based immune signaling, glucosylates N-hydroxypipecolic acid and controls basal pathogen defense

Glucosylation modulates the biological activity of small molecules and frequently leads to their inactivation. The Arabidopsis thaliana glucosyltransferase UGT76B1 is involved in conjugating the stress hormone salicylic acid (SA) as well as isoleucic acid (ILA). Here, we show that UGT76B1 also glucosylates N-hydroxypipecolic acid (NHP), which is synthesized by FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) and activates systemic acquired resistance (SAR). Upon pathogen attack, Arabidopsis leaves accumulate two distinct NHP hexose conjugates, NHP-O-{beta}-glucoside and NHP glucose ester, which are oppositely regulated by SA. ugt76b1 mutants specifically fail to generate the NHP-O-{beta}-glucoside, and recombinant UGT76B1 synthesizes NHP-O-{beta}-glucoside in vitro in competition with SA and ILA. The loss of UGT76B1 elevates the endogenous levels of NHP in addition to SA and ILA and establishes a SAR-like, primed immune status without pathogen infestation. The introgression of the fmo1 background lacking NHP biosynthesis into ugt76b1 abolishes the SAR-like resistance phenotype indicating an important function of UGT76B1-mediated NHP glucosylation in balancing the defense status. Our results further indicate that ILA promotes and SA finally executes the NHP-triggered immunity via the glucosyltransferase UGT76B1 as the common metabolic hub. Thus, UGT76B1 controls the levels of active NHP, SA, and ILA in concert to modulate plant immune signaling.

plant biology↗

bioRxiv2026

Impact of Solvent Extraction on Bioactive Properties of Lavandula x intermedia: Antibacterial, Antioxidant and Phenolic Content Analysis

Lavandula x intermedia (lavandin) is an economically important hybrid valued for its high essential oil yield; however, studies linking solvent polarity to its bioactivity and phenolic composition remain limited. This study evaluated the effects of nine extraction solvents on the antibacterial and antioxidant activities of L. x intermedia, determined total phenolic content (TPC), and examined the relationship between chemical composition and biological activity. Aerial parts were extracted by maceration. Antibacterial activity was assessed using the broth microdilution method against four bacterial strains, while antioxidant capacity was measured by the DPPH assay. TPC was determined using the Folin-Ciocalteu method, and phenolic compounds were analyzed via LC-MS/MS. Results showed that solvent polarity significantly influenced bioactivity. Diethyl ether extracts exhibited the highest TPC (267.65 mg GAE/g), strongest antioxidant activity (IC50: 72.26 {micro}g/mL), and notable antibacterial effects (MIC: 125 {micro}g/mL), especially against Gram-positive bacteria. A strong positive correlation (r = 0.887) was observed between phenolic content and antimicrobial activity. LC-MS/MS identified key compounds, including fumaric acid, resveratrol, and hydroxycinnamic acid. Overall, moderate-polarity solvents such as diethyl ether and methanol were most effective, highlighting L. x intermedia as a promising natural source for pharmaceutical and nutraceutical applications.

plant biology↗

bioRxiv2025

Chloroplast-mitochondria synergy modulates responses to iron limitation in two Thalassiosira diatom species

Iron is naturally present at low levels in modern oceans, but it remains essential for marine life. Ocean-dwelling organisms such as oceanic phytoplankton must therefore adapt to the available levels. Among phytoplankton, diatoms are a highly diverse and successful taxon that includes the Thalassiosira genus. As a group, diatoms contribute around 20% of global primary photosynthetic productivity, they have also developed specific resources allowing them to thrive in low-iron regions. However, the major biological factors underlying their success in these ocean environments remain unknown. Here, we compared two Thalassiosira species: T. oceanica from iron-poor open-ocean; and T. pseudonana, from iron-rich coastal waters. Since iron is essential for both photosynthesis and respiration, we examined the specificities of the bioenergetic machineries in these organisms using a combination of photo-physiological, proteomics, and FIB-SEM methods. We particularly focused on chloroplast-mitochondrial coupling, a mechanism deployed by diatoms to ensure optimal transfer of photosynthetic products to promote cell growth. This study of this mechanism in the context of iron limitation reveals that the two diatoms differentially remodel chloroplast compartments in response to iron limitation. Their tolerance to these conditions is also linked to distinct constitutive mitochondrial architectures.

plant biology↗

bioRxiv2026

Comparative reproductive biology and impacts of selfing in two epiphytic Bromeliaceae

AO_SCPLOWBSTRACTC_SCPLOWThe breeding systems of most Bromeliaceae have not been tested directly. We characterized the floral biology and breeding systems of two epiphytic, tank-forming bromeliads endemic to the Atlantic Forest of Brazil, Vriesea rafaelii (Tillandsioideae) and Billbergia brasiliensis (Bromelioideae), using controlled self- and cross-pollinations in a greenhouse. Both species were self-compatible. Based on seed-set, the self-compatibility index was 0.59 in V. rafaelii and 0.87 in B. brasiliensis. In B. brasiliensis, selfed flowers produced lighter fruits and lighter seeds than outcrossed flowers, whereas in V. rafaelii fruit and seed mass did not differ between self- and cross-pollinated flowers. A subset of V. rafaelii flowers showed a spatial separation of the anthers and stigma (herkogamy) that prevented autonomous selfing. These results add two species to the small set of bromeliads with experimentally characterized breeding systems and highlight differences in reproductive output with selfing.

plant biology↗

bioRxiv2021

Explainable machine learning models of major crop traits from satellite-monitored continent-wide field trial data

Four species of grass generate half of all human-consumed calories1. However, abundant biological data on species that produce our food remains largely inaccessible, imposing direct barriers to understanding crop yield and fitness traits. Here, we assemble and analyse a continent-wide database of field experiments spanning ten years and hundreds of thousands of machine-phenotyped populations of ten major crop species. Training an ensemble of machine learning models, using thousands of variables capturing weather, ground-sensor, soil, chemical and fertiliser dosage, management, and satellite data, produces robust cross-continent yield models exceeding R2 = 0.8 prediction accuracy. In contrast to black box analytics, detailed interrogation of these models reveals fundamental drivers of crop behaviour and complex interactions predicting yield and agronomic traits. These results demonstrate the capacity of machine learning models to build unified, interpretable, and explainable models of crop behaviour, and highlight the powerful role of data in the future of food.

plant biology↗

bioRxiv2021

Autophagy promotes photomorphogenesis during seedling development in Arabidopsis in carbon limiting conditions

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation condition and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12h post illumination. We investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in photomorphogenesis. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected photomorphogenesis. Moreover, we showed that lower levels of carbon resources exacerbated the delay in photomorphogenesis whereas higher levels of carbon resources had an opposite effect. This work provides evidence that autophagic process operate during de-etiolation in a manner that contributes to photomorphogenesis through increasing lipid turnover to physically or energetically sustain photomorphogenesis.

plant biology↗

bioRxiv2018

Can you make morphometrics work when you know the right answer? Pick and mix approaches for apple identification.

Morphological classification of living things has challenged science for several centuries and has led to a wide range of objective morphometric approaches in data gathering and analysis. In this paper we explore those methods using apple cultivars, a model biological system in which discrete groups are pre-defined but in which there is a high level of overall morphological similarity. The effectiveness of morphometric techniques in discovering the groups is evaluated using statistical learning tools. No one technique proved optimal in classification on every occasion, linear morphometric techniques slightly out-performing geometric (72.6% accuracy on test set versus 66.7%). The combined use of these techniques with post-hoc knowledge of their individual successes with particular cultivars achieves a notably higher classification accuracy (77.8%). From this we conclude that even with pre-determined discrete categories, a range of approaches is needed where those categories are intrinsically similar to each other, and we raise the question of whether in studies where potentially continuous natural variation is being categorised the level of match between categories is routinely set too high.

plant biology↗

bioRxiv2026

FLASH-P: Turning decades of biology into accurate causal networks with AI agents

Mechanistic networks that encode causal regulatory logic can predict the effects of genetic and environmental perturbations but constructing them is a bottleneck in systems biology because the relevant knowledge lies scattered across thousands of resources, untapped for both building and validating such networks. Here we present FLASH-P, a multi-agent framework that autonomously curates this literature into perturbable, signed-directed network models for any trait-species combination in under an hour without much computational power. Twelve FLASH-P networks across seven species predicted the directional outcome of 1,088 published perturbations with a mean accuracy of 90%. This accuracy was driven by the regulatory topology FLASH-P constructs, which is why it outperformed knowledge-graph derived networks. Its merging agent combined six networks into one that preserved single-trait accuracy and recovered pleiotropic effects, and consolidated independent runs of one trait into a comprehensive, high-accuracy network. FLASH-P networks enable applications that require trait models.

Plant Biology↗

bioRxiv2024

Partial redundancy buffers deleterious effects of mutating DNA methyltransferase 1-1 (MET1-1) in polyploid wheat

DNA methylation is conserved across biological kingdoms, playing important roles in gene expression, transposable element silencing and genome stability. Altering DNA methylation could generate additional phenotypic variation for crop breeding, however the lethality of epigenetic mutants in crop species has hindered its investigation. Here, we exploit partial redundancy between homoeologs in polyploid wheat to generate viable mutants in the DNA methyltransferase 1-1 (MET1-1) gene with altered methylation profiles. In both Triticum turgidum (tetraploid wheat) and Triticum aestivum (hexaploid wheat) we identified clear segregation distortions of higher-order mutants (5/6 and 6/6 mutant met1-1 copies in hexaploid and 3/4 and 4/4 copies in tetraploid) when genotyping segregating seeds and seedlings, which we attribute to reduced transmission of null mutant gametes. We found that the reduced transmission occurred from both the maternal and paternal gametes, however, we did not detect any deleterious effects on pollen development. The loss of four or more functional copies of MET1-1 results in decreased CG methylation in hexaploid wheat. Changes to gene expression increase stepwise with the number of mutant alleles suggesting a dosage dependent effect. Finally, we identify heritable changes to flowering and awn phenotypes which segregate independently of MET1-1. Together our results demonstrate that polyploidy can be leveraged to generate quantitative changes to CG methylation without the lethal consequences observed in other crops, opening the potential to exploit novel epialleles in plant breeding.

plant biology↗

bioRxiv2022

INCREASING DIVERSITY AMONG LENS SPECIES FOR IMPROVING BIOLOGICAL NITROGEN FIXATION IN LENTIL

Exotic germplasm is a key resource for reintroducing genetic variability into cultivars. We evaluated 36 accessions from cultivated lentil (Lens culinaris Medik.) and six related wild species, inoculated with a commercial strain of Rhizobium leguminosarum bv. viciae under greenhouse conditions. The objective was to explore Lens species and/or accessions that could contribute higher biological nitrogen fixation (BNF) ability to the lentil crop. A split plot design was used with either Rhizobium inoculation, added Nitrogen (N) or neither, as the main plots, and accessions in subplots randomized in blocks. Two repeats of the experiment were evaluated at flowering for N fixation and nodulation characters, and two subsequent experiments, with a subset of 14 accessions, were evaluated at maturity for seed production, seed quality and harvest index. Differences in phenotypic expression did not correspond to any particular Lens species. CDC Greenstar exhibited some of the highest N fixation values observed among the cultivars and also superior yield results compared to the added N treatment. Wild accessions, including IG 72643 (L. orientalis), displayed unique and multiple desirable characteristics compared to cultivars including indeterminate nodulation, higher N translocation, stable yield compared to added N treatment and exceptionally high protein concentration in seeds.

plant biology↗

bioRxiv2015

GLO-Roots: an imaging platform enabling multidimensional characterization of soil-grown roots systems

Root systems develop different root types that individually sense cues from their local environment and integrate this information with systemic signals. This complex multidimensional amalgam of inputs enables continuous adjustment of root growth rates, direction and metabolic activity that define a dynamic physical network. Current methods for analyzing root biology balance physiological relevance with imaging capability. To bridge this divide, we developed an integrated imaging system called Growth and Luminescence Observatory for Roots (GLO-Roots) that uses luminescence-based reporters to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. We have developed image analysis algorithms that allow the spatial integration of soil properties, gene expression and root system architecture traits. We propose GLO-Roots as a system that has great utility in presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for studying the multi-dimensional nature of such processes.

Plant Biology↗

bioRxiv2026

Identifying water stress response haplotypes in barley using latent environmental covariates

PurposeGenotype-by-environment (G x E) interactions represent a major obstacle to increasing genetic gain in crop breeding, with the underlying physiological drivers often remaining obscured within conventional statistical models. This case study presents a novel framework that transforms the latent factors from Factor Analytic (FA) multi-environment trial (MET) models into heritable quantitative traits, enabling the genetic dissection of adaptive response patterns. MethodsA Factor Analytical Linear Mixed Model (FA-LMM) was fit to plot-level yield data for 1,036 barley genotypes across eight Australian trials. ResultsCorrelation of the factor loadings with APSIM-simulated environmental covariates demonstrated that the second latent factor FA2 was strongly correlated with the Water Stress Index (r = -0.83) during the critical flowering period, establishing water availability as the main biological axis of crossover Gx E. Genotypic scores for the derived traits, Overall Performance (OP) and Water Stress Response (WSR), were subjected to high-resolution haplotype-based mapping using local Genomic Estimated Breeding Values (GEBV). ConclusionThis analysis successfully identified major genomic regions that accounted for a substantial proportion of the additive genetic variance. Gene Ontology enrichment of candidate genes within the top haploblocks implicated fundamental pathways related to energy homeostasis, root development, and stress response, with notable candidates including FTsH11, BPS1, and TDP1. The distribution of favourable Haplotypes of Interest (HOI) in elite cultivars suggested a historical signature of inadvertent selection for these adaptive mechanisms. This framework provides an explicit bridge between statistical modelling and functional genomics, offering breeders actionable genetic targets for accelerated development of climate-resilient cereals.

plant biology↗

bioRxiv2026

PEELING WALLS1 encodes a GT106 protein required for seed surface integrity

Rhamnogalacturonan I (RG-I) is a major pectin domain and an important wall component for cell-cell adhesion. Arabidopsis seed mucilage serves as a powerful model to identify and characterize pectin-related enzymes, since this gelatinous capsule is composed predominantly of RG-I. Although multiple glycosyltransferase (GT) families participate in pectin biosynthesis, only a limited number of their members have been functionally characterized in vivo or biochemically characterized in vitro. In this study, we characterized the biological functions of PEELING WALLS1 (PEEL1), a Golgi-localized GT106 protein that is related to known RG-I rhamnosyltransferases (RRTs). Knocking out PEEL1, but not its close paralog PEEL1-LIKE (PEEL1L), led to patchy mucilage release from seeds upon hydration. However, RG-I content was not compromised in mucilage extracted from peel1 or peel1 peel1L mutant seeds. Treatment of seeds with a cation chelator restored mucilage expansion but failed to rescue the underlying defect in epidermal cell adhesion. Histological experiments and transgene complementation demonstrated that optimal expression of PEEL1 is required for the adhesion of primary walls to the seed surface. Collectively, these findings identify PEEL1 as a previously unrecognized contributor to primary cell wall adhesion and suggest that different RRT-related proteins contribute to unique pectic structures or localizations in vivo, rather than total mucilage RG-I abundance.

plant biology↗

bioRxiv2020

In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5 end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5 UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

plant biology↗

bioRxiv2021

Nonadditive gene expression is correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow (Salix spp.)

Many studies have highlighted the complex and diverse basis for heterosis in inbred crops. Despite the lack of a consensus model, it is vital that we turn our attention to understanding heterosis in undomesticated, heterozygous, and polyploid species, such as willow (Salix spp.). Shrub willow is a dedicated energy crop bred to be fast-growing and high yielding on marginal land without competing with food crops. A trend in willow breeding is the consistent pattern of heterosis in triploids produced from crosses between diploid and tetraploid species. Here, we test whether differentially expressed genes are associated with heterosis in triploid families derived from diploid S. purpurea, diploid S. viminalis, and tetraploid S. miyabeana parents. Three biological replicates of shoot tips from all family progeny and parents were collected after 12 weeks in the greenhouse and RNA extracted for RNA-Seq analysis. This study provides evidence that nonadditive patterns of gene expression are correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow. Expression-level dominance was most correlated with heterosis for biomass yield traits and was highly enriched for processes involved in starch and sucrose metabolism. In addition, there was a global dosage effect of parent alleles in triploid hybrids, with expression proportional to copy number variation. Importantly, differentially expressed genes between family parents were most predictive of heterosis for both field and greenhouse collected traits. Altogether, these data will be used to progress models of heterosis to complement the growing genomic resources available for the improvement of heterozygous perennial bioenergy crops.

plant biology↗

bioRxiv2020

Characterization of the Cannabis sativa glandular trichome proteome

Cannabis sativa has been cultivated since antiquity as a source of fibre, food and medicine. The recent resurgence of Cannabis as a cash crop is mainly driven by the medicinal and therapeutic properties of its resin, which contains compounds that interact with the human endocannabinoid system. Compared to other medicinal crops of similar value, however, little is known about the biology of C. sativa. Glandular trichomes are small hair-like projections made up of stalk and head tissue and are responsible for the production of the resin in C. sativa. Trichome productivity, as determined by Cannabis sativa resin yield and composition, is only beginning to be understood at the molecular level. In this study the proteomes of glandular trichome stalks and heads, were investigated and compared to the proteome of the whole flower tissue, to help elucidate Cannabis sativa glandular trichome biochemistry. The data suggested that the floral tissue acts as a major source of carbon and energy to the glandular trichome head sink tissue, supplying sugars which drive secondary metabolite biosynthesis in the glandular trichome head; the location of the secretory cells. The trichome stalk seems to play only a limited role in secondary metabolism and acts as both source and sink.

plant biology↗

bioRxiv2022

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and lipid biosynthesis capacity

Mitochondria are often considered the power stations of the cell, playing critical roles in various biological processes such as cellular respiration, photosynthesis, stress responses and programmed cell death. To maintain the structural and functional integrities of mitochondria, it is crucial to achieve a defined membrane lipid composition between different lipid classes wherein specific proportions of individual lipid species are present. Although mitochondria are capable of self-synthesizing a few lipid classes, many phospholipids are synthesized in the endoplasmic reticulum and transferred to mitochondria via membrane contact sites, as mitochondria are excluded from the vesicular transportation pathway. However, knowledge on the capability of lipid biosynthesis in mitochondria and the precise mechanism of maintaining the homeostasis of mitochondrial lipids is still scarce. Here we describe the lipidome of mitochondria isolated from Arabidopsis leaves, including the molecular species of glycerolipids, sphingolipids and sterols to depict the lipid landscape of mitochondrial membranes. In addition, we define proteins involved in lipid metabolism by proteomic analysis and compare our data with mitochondria from cell cultures since they still serve as model system. Proteins putatively localized to the membrane contact sites are proposed based on the proteomic results and online databases. Collectively, our results suggest that leaf mitochondria are capable - with the assistance of membrane contact site-localised proteins - of generating several lipid classes including phosphatidylethanolamines, cardiolipins, diacylgalactosylglycerols and free sterols. We anticipate our work to be a foundation to further investigate the functional roles of lipids and their involvement in biochemical reactions in plant mitochondria. One sentence summaryThe lipid landscape of plant mitochondria suggests that they are capable in generating several phospholipid classes with the assistant of membrane contact site-localized proteins.

plant biology↗