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Results for “plant biology”

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bioRxiv2017

Comparative Qualitative Phosphoproteomics Analysis Identifies Shared Phosphorylation Motifs and Associated Biological Processes in Flowering Plants

Phosphorylation is regarded as one of the most prevalent post-translational modifications and plays a key role in regulating cellular processes. In this work we carried out a comparative bioinformatics analysis of phosphoproteomics data, to profile two model species representing the largest subclasses in flowering plants the dicot Arabidopsis thaliana and the monocot Oryza sativa, to understand the extent to which phosphorylation signaling and function is conserved across evolutionary divergent plants. Using pre-existing mass spectrometry phosphoproteomics datasets and bioinformatic tools and resources, we identified 6,537 phosphopeptides from 3,189 phosphoproteins in Arabidopsis and 2,307 phosphopeptides from 1,613 phosphoproteins in rice. The relative abundance ratio of serine, threonine, and tyrosine phosphorylation sites in rice and Arabidopsis were highly similar: 88.3: 11.4: 0.4 and 86.7: 12.8: 0.5, respectively. Tyrosine phosphorylation shows features different from serine and threonine phosphorylation and was found to be more frequent in doubly-phosphorylated peptides in Arabidopsis. We identified phosphorylation sequence motifs in the two species to explore the similarities, finding nineteen pS motifs and two pT motifs that are shared in rice and Arabidopsis; among them are five novel motifs that have not previously been described in both species. The majority of shared motif-containing proteins were mapped to the same biological processes with similar patterns of fold enrichment, indicating high functional conservation. We also identified shared patterns of crosstalk between phosphoserines with motifs pSXpS, pSXXpS and pSXXXpS, where X is any amino acid, in both species indicating this is an evolutionary conserved signaling mechanism in flowering plants. However, our results are suggestive that there is greater co-occurrence of crosstalk between phosphorylation sites in Arabidopsis, and we were able to identify several pairs of motifs that are statistically significantly enriched to co-occur in Arabidopsis proteins, but not in rice.

bioinformatics↗

bioRxiv2017

Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a \"peripheral infrastructure\" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

plant biology↗

bioRxiv2026

A molecular description of plant cellulose biosynthesis inhibition

Dictating cell growth and morphology, cellulose biosynthesis is intrinsic to plant cell biology. Accordingly, cellulose biosynthesis inhibitors (CBIs) are important herbicides, toxins, and experimental tools. We currently lack mechanistic understanding of CBI activity, preventing engineering of herbicide selectivity and disease immunity. Contrasting classical inhibitors, we unexpectedly identify the unusual Streptomyces phytotoxin thaxtomin A as the only in vitro-active CBI, with unprecedentedly broad-spectrum activity against various cellulose synthase enzymes. High-resolution cryo-electron microscopy reveals that thaxtomin A leverages exotic nitroaromatic chemistry to target a strictly conserved site in cellulose synthases polysaccharide secretion channel. Strikingly, in vitro biosynthesis and biophysical assays demonstrate thaxtomin As near-picomolar efficacy. Plant and algal systems reveal that its global arrest of cellulose biosynthesis produces an osmotically driven crisis in expanding cells. Finally, site-directed mutagenesis generates the first toxin-resistant cellulose synthase. Our results underscore celluloses critical function in plant lifeforms and inform efforts to inhibit related enzymes across kingdoms.

plant biology↗

bioRxiv2024

Ohmic analogies and metaphorical circuits: Vascular partitioning of leaf air spaces and stomatal patchiness can create apparent undersaturation and gradient inversion.

RationaleAnalyses of leaf gas exchange rely on an Ohmic analogy that arrays single stomatal, internal air space, and mesophyll conductances in series. Such models underlie inferences of mesophyll conductance and the relative humidity of leaf airspaces, reported to fall as low as 80%. An unresolved question is whether such Ohmic models are biased with respect to real leaves, whose internal air spaces are chambered at various scales by vasculature. DescriptionTo test whether undersaturation could emerge from modeling artifacts, we compared Ohmic model estimates with true parameter values for a chambered leaf with varying distributions and magnitudes of leaf surface conductance ("patchiness"). Key ResultsDistributions of surface conductance can create large biases in gas exchange calculations. Both apparent unsaturation and internal CO2 gradient inversion can be produced by the evolution of particular distributions of stomatal apertures consistent with a decrease in surface conductance, as might occur under increasing vapor pressure deficit. Main conclusionIn gas exchange experiments, the behaviors of derived quantities defined by simple Ohmic models are highly sensitive to the true partitioning of flux and stomatal apertures across leaf surfaces. We need new methods to disentangle model artifacts from real biological responses.

plant biology↗

bioRxiv2018

Molecular characterization of beast’s maguey (Agave antillarum Descourt), in the Dominican Republic

BackgroundThis research project was proposed to determine the genetic variability (if any) of beast maguey (Agave antillanarum Decourt) populations, by means of molecular markers of the AFLPs (Amplified Fragment Length Polymorphism) type. For the determination of genetic variability, samples were collected from wild populations of maguey growing in some regions of the Dominican Republic, mainly the north, northwest, south and southwest regions, using statistical techniques already established for these cases. These samples were processed and analyzed in the plant molecular biology laboratory of the Centtro de Biotecnologia Vegetal (CEBIVE) of Instituto de Innovacion en Biotecnologia e Industria (IIBI) in Santo Domingo, Dominican Republic.\n\nResultsAs for the genetic diversity is concerned, it was found that several of the populations studied differ from each other without taking into account the geographic distance where they grow. We concluded that there is a possibility that more than one species of Agave antillarun could be found in the wild in the Dominican Republic.

plant biology↗

bioRxiv2020

Reconstructing the history and biological consequences of a plant invasion on the Galapagos islands

The introduction of non-native species into new habitats is one of the foremost risks to global biodiversity. Here, we evaluate a recent invasion of wild tomato (Solanum pimpinellifolium) onto the Galapagos islands from a population genomic perspective, using a large panel of novel collections from the archipelago as well as historical accessions from mainland Ecuador and Peru. We infer a recent invasion of S. pimpinellifolium on the islands, largely the result of a single event from central Ecuador which, despite its recency, has rapidly spread onto several islands in the Galapagos. By reconstructing patterns of local ancestry throughout the genomes of invasive plants, we uncover evidence for recent hybridization and introgression between S. pimpinellifolium and the closely related endemic species Solanum cheesmaniae. Two large introgressed regions overlap with known fruit color loci involved in carotenoid biosynthesis. Instead of red fruits, admixed individuals with endemic haplotypes at these loci have orange fruit colors that are typically characteristic of the endemic species. We therefore infer that introgression explains the observed trait convergence. Moreover, we infer roles for two independent loci in driving this pattern, and a likely history of selection favoring the repeated phenotypic transition from red to orange fruits. Together, our data reconstruct a complex history of invasion, expansion, and gene flow among wild tomatoes on the Galapagos islands. These findings provide critical data on the evolutionary importance of hybridization during colonization and its role in influencing conservation outcomes. Significance StatementThe isolation and unique diversity of the Galapagos Islands provide numerous natural experiments that have enriched our understanding of evolutionary biology. Here we use population genomic sequencing to reconstruct the timing, path, and consequences of a biological invasion by wild tomato onto the Galapagos. We infer that invasive populations originated from a recent human-mediated migration event from central Ecuador. Our data also indicate that invasive populations are hybridizing with endemic populations, and that this has led to some invasive individuals adopting both fruit color genes and the fruit color characteristic of the endemic island species. Our results demonstrate how hybridization can shape patterns of trait evolution over very short time scales, and characterize genetic factors underlying invasive success.

evolutionary biology↗

bioRxiv2019

A theophylline-responsive riboswitch regulates expression of nuclear-encoded genes in Arabidopsis

Ligand-responsive synthetic riboswitches are versatile and innovative tools for external gene regulation in pro- and eukaryotes. Riboswitches are small cis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genes in planta has not been reported so far. Here we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3-UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent downregulation of the Green Fluorescent Protein (GFP) reporter gene in a dose- and time- dependent manner. Insertion of the riboswitch into the One Helix Protein 1 (OHP1) gene allowed complementation of ohp1 mutants and induction of the mutant phenotype by theophylline. GFP or OHP1 transcript levels were downregulated by maximally 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.\n\nOne sentence summaryArtificial, ligand-responsive RNA aptazymes are an efficient tool for dose- and time-dependent external control of nuclear gene expression in plants.

plant biology↗

bioRxiv2023

Spatial transcriptomic atlas of shoot organogenesis in tomato callus

Callus is a reprogrammed transitional cell mass during plant regeneration. Pluripotent callus cells develop into fertile shoots through de novo shoot organogenesis. This study represents a pioneering effort in exploring the spatial transcriptome of tomato callus during shoot regeneration, using technologies including BGI Stereo-seq, BMKMANU S1000, and 10x Visium. The results indicate that the callus comprises highly heterogeneous cells, classified into various cell types based on spatial gene expression and histological observation, including epidermis, shoot primordium, vascular tissue, inner callus, and outgrowth shoots. The developmental trajectories from shoot primordium to outgrowth shoot are traced, and vascular tissue development is characterized. The single-cell resolution spatial approach reveals the origin of shoot primordia from the sub-epidermis. The spatial full length RNA sequencing shows high incompletely spliced (IS) ratios in the shoot primordium cells. These findings enhance our knowledge of plant organogenesis and highlight the significance of spatial biology in plant research.

cell biology↗

bioRxiv2026

A single-cell transcriptome atlas of the barley root apical meristem uncovers conserved and divergent roles of HvWOX5

Single-cell approaches have transformed plant developmental biology; however, cell-type-resolved resources for cereals remain limited. Here, we present a single-cell transcriptome atlas of the barley root apical meristem (RAM), which resolves 24 transcriptionally distinct cell populations. We assigned major root cell identities by integrating marker gene validation using Hybridization Chain Reaction (HCR) RNA fluorescence in situ hybridization and spatial transcriptomics with cross-species comparisons of published root atlases. Pseudotime analysis reconstructed developmental trajectories from the quiescent center to differentiating tissues, supporting the spatial and developmental organization of the atlas. We further demonstrated the utility of this resource by identifying HvWOX5 expression in the quiescent center and metaxylem and showing that HvWOX5 loss-of-function mutants displayed reduced root and meristem lengths, altered stem cell niche homeostasis, and disrupted metaxylem organization. Taken together, this atlas provides a framework for dissecting barley root development and identifies HvWOX5 as a key regulator of RAM organization and metaxylem patterning.

Plant Biology↗

bioRxiv2016

Fungal Induced Protein Hyperacetylation Identified by Acetylome Profiling

Lysine acetylation is a key post-translational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established and it is known that pathogen effector proteins encoding acetyltransferses can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here we investigate how the effector molecule HC-toxin, a histone deacetylase inhibitor, produced by Cochliobolus carbonum race 1 promotes pathogen virulence in maize through altering protein acetylation. Using mass spectrometry we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HC-toxin as well as HC-toxin deficient or producing strains of C. carbonum. Analyses of these data demonstrate that acetylation is a widespread post-translational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HC-toxin enabled us to show that the activity of plant-encoded enzymes can be modulated to alter acetylation of non-histone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function and offer insight into the complex immune response triggered by virulent C. carbonum.

Plant Biology↗

bioRxiv2022

Apiaceae FNS I originated from F3H through tandem gene duplication

BackgroundFlavonoids are specialized metabolites with numerous biological functions in stress response and reproduction of plants. Flavones are one subgroup that is produced by the flavone synthase (FNS). Two distinct enzyme families evolved that can catalyze the biosynthesis of flavones. While the membrane-bound FNS II is widely distributed in seed plants, one lineage of soluble FNS I appeared to be unique to Apiaceae species. ResultsWe show through phylogenetic and comparative genomic analyses that Apiaceae FNS I evolved through tandem gene duplication of flavanone 3-hydroxylase (F3H) followed by neofunctionalization. Currently available datasets suggest that this event happened within the Apiaceae in a common ancestor of Daucus carota and Apium graveolens. The results also support previous findings that FNS I in the Apiaceae evolved independent of FNS I in other plant species. ConclusionWe validated a long standing hypothesis about the evolution of Apiaceae FNS I and predicted the phylogenetic position of this event. Our results explain how an Apiaceae-specific FNS I lineage evolved and confirm independence from other FNS I lineages reported in non-Apiaceae species.

plant biology↗

bioRxiv2022

Variation in morpho-physiological and metabolic responses to low nitrogen stress across the sorghum association panel

Access to biologically available nitrogen is a key constraint on plant growth in both natural and agricultural settings. Variation in tolerance to nitrogen deficit stress and productivity in nitrogen limited conditions exists both within and between plant species. Here we quantified variation in the metabolic, physiological, and morphological responses of a sorghum association panel assembled to represent global genetic diversity to long term, moderate, nitrogen deficit stress and the relationship of these responses to grain yield under both conditions. Grain yield exhibits substantial genotype by environment interaction while many other morphological and physiological traits exhibited consistent responses to nitrogen stress across the population. Large scale nontargeted metabolic profiling for a subset of lines in both conditions identified a range of metabolic responses to long term nitrogen deficit stress as well as several metabolites associated with variation in the degree of yield plasticity specific sorghum genotypes exhibited in response to nitrogen deficit stress.

plant biology↗

bioRxiv2025

A Simple and Scalable Chopped-Thallus Transformation Method for Marchantia polymorpha

The liverwort Marchantia polymorpha has emerged as a valuable model for studying fundamental biological processes and the evolutionary history of land plants. Agrobacterium-mediated transformation is widely used for genetic modification of M. polymorpha using spores, thalli, and gemmae. While spores offer high transformation efficiency, they result in diverse genetic backgrounds due to sexual reproduction. Conversely, thallus- and gemma-based methods maintain genetic consistency but are impractical for large-scale applications. To address these limitations, we developed a novel chopped-thallus transformation method. This technique involves generating numerous plant fragments by chopping thalli, eliminating the need for complex preprocessing. The method demonstrated superior transformation efficiency compared to traditional approaches and achieved sufficient numbers of transformants using simplified Gamborgs B5 medium, previously considered suboptimal. This scalable and straightforward method enables the generation of large numbers of genetically consistent transformants, facilitating high-throughput experiments, including mutant screening and other large-scale applications.

plant biology↗

bioRxiv2025

Floral innovation through modifications in stem cell peptide signaling.

Understanding how evolution shapes genetic networks to create new developmental forms is a central question in biology. Asteraceae (sunflower family) comprise 10% of flowering plants and have capitula, a novel flowering shoot (inflorescence) that mimics a single flower (1, 2). During capitulum development, shoot stem cells undergo prolonged proliferation relative to other species (3, 4). Here we show that capitulum evolution paralleled decreases in CLAVATA3 peptide (CLV3p) signaling, a conserved repressor of stem cell proliferation. Asteraceae CLV3p displays reduced receptor binding and downstream transcriptional outputs. Reversion of CLV3 to a more active form impairs Asteraceae stem cell regulation and capitulum development. Lastly, we trace CLV3 evolution across the Asterales allowing inferences on capitulum evolution. Our findings reveal novel evolutionary mechanisms in plant reproduction and suggest approaches for engineering crops.

plant biology↗

bioRxiv2018

Fragmentation of pooled PCR products for highly multiplexed TILLING

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING, where whole genome approaches become cost prohibitive. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multidimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Thirty-two PCR products were produced from genomic DNA pools representing 265 pooled barley mutant lines. Mutant lines were produced with the chemical mutagen ethyl methanesulfonate. Samples were subjected to 2x300PE illumina sequencing. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach.

plant biology↗

bioRxiv2020

Control of Vein-Forming, Striped Gene-Expression by Auxin Signaling

Activation of gene expression in striped domains is a key building block of biological patterning, from the recursive formation of veins in plant leaves to that of ribs and vertebrae in our bodies. In animals, gene expression is activated in striped domains by the differential affinity of broadly expressed transcription factors for their target genes and the combinatorial interaction between such target genes. In plants, how gene expression is activated in striped domains is instead unknown. We address this question for the broadly expressed MONOPTEROS (MP) transcription factor and its target gene ARABIDOPSIS THALIANA HOMEOBOX FACTOR8 (ATHB8). We find that ATHB8 promotes vein formation and that such vein-forming function depends on both levels of ATHB8 expression and width of ATHB8 expression domains. We further find that ATHB8 expression is activated in striped domains by a combination of (1) activation of ATHB8 expression through binding of peak levels of MP to a low-affinity MP-binding site in the ATHB8 promoter and (2) repression of ATHB8 expression by MP target genes of the INDOLE-3-ACETIC-ACID-INDUCIBLE family such as BODENLOS. Our findings suggest that a common regulatory logic controls activation of gene expression in striped domains in both plants and animals despite the independent evolution of their multicellularity.

plant biology↗

bioRxiv2023

Genome size is positively correlated with extinction risk in herbaceous angiosperms

O_LIAngiosperms with large genomes experience nuclear-, cellular- and organism-level constraints that may limit their phenotypic plasticity and ecological niche. These constraints have been documented to vary across lineages, life-history strategies, ecogeographic patterns and environmental conditions. Therefore, we test the hypotheses that extinction risk is higher in large-genomed compared to small-genomed species, and that the effect of genome size varies across three selected covariates: life form, endemism, and climatic zones. C_LIO_LIWe collated genome size and extinction risk information for a representative sample of angiosperms comprising 3,250 species, which we analyzed alongside life form, endemism and climate variables using a phylogenetic framework. C_LIO_LIAngiosperm genome size is positively correlated with extinction risk, a pattern driven by a signal in herbaceous but not woody species, regardless of climate and endemism. The influence of genome size is stronger in endemic herbaceous species, but is relatively homogenous across different climates. Beyond its indirect link via endemism and climate, genome size also influences extinction risk directly and significantly. C_LIO_LIGenome size may serve as a proxy for difficult-to-measure parameters associated with resilience and vulnerability in herbaceous angiosperms. Therefore, it merits further exploration as a useful biological attribute for understanding intrinsic extinction risk and augmenting plant conservation efforts. C_LI

plant biology↗

bioRxiv2024

Hydrothermal pretreatment renders peat susceptible to enzymatic saccharification

Sphagnum peat bogs store a large fraction of biologically-bound carbon, due to a steady accumulation of plant material over millennia. The resistance of Sphagnum biomass to decay is poorly understood but of high importance for preservation efforts and climate models. It is shown that peat cellulose and other glucose-rich polysaccharides are readily degradable by a commercial enzyme cocktail designed for the industrial saccharification of lignocellulose of vascular plants. However, prior hydrothermal pretreatment of peat was required for the enzymes to gain access to the polysaccharides. The pretreatment itself released monosaccharides and glucose-containing soluble oligosaccharides. The monosaccharide profile released from hydrothermal pretreatment was consistent with the expected hemicellulose content of Sphagnum and was clearly different from that seen for pretreated tissue from vascular plants, such as pretreated wheat straw. Cellulose retained in the insoluble part of peat was cleaved at a similar or higher rate compared to cellulose from vascular plant tissues. Confocal laser scanning microscopy showed that the hydrothermal pretreatment disrupted the cells and relocated lignin-like compounds. Peat contains a high concentration of iron, which likely explains the pronounced acidification observed for the pretreated peat slurry at ambient conditions during saccharification assays. The acidification is due to abiotic oxidative reactions that also inactivate the enzymes. Adding catalase to the reactions alleviated enzyme inactivation and essentially stopped acidification during saccharification. This study confirms the importance of considering those abiotic oxidative reactions that take place in drained peat material.

plant biology↗