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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Mild drought induces phenotypic and DNA methylation plasticity but no transgenerational effects in Arabidopsis

Whether environmentally induced changes in phenotypes can be heritable is a topic with revived interest, in part because of observations in plants that heritable trait variation can occur without DNA sequence mutations. This other system of inheritance, called transgenerational epigenetics, typically involves differences in DNA methylation that are stable across multiple generations. However, it remains unknown if such a system responds to environmental changes and if it could therefore provide a rapid way for plants to generate adaptive heritable phenotypic variation. Here, we used a well-controlled phenotyping platform and whole-genome bisulfite sequencing to investigate potential heritable effects of mild drought applied over two successive generations in Arabidopsis thaliana. Plastic phenotypic responses were observed in plants exposed to drought. After an intervening generation without stress, descendants of stressed and non-stressed plants were phenotypically indistinguishable, except for very few trait-based parental effects, and irrespective of whether they were grown in control conditions or under water deficit. Moreover, while mild drought induced changes to the DNA methylome of exposed plants, DNA methylation variants were not inherited. These findings add to the growing body of evidence indicating that transgenerational epigenetics is not a common response of plants to environmental changes.

plant biology↗

bioRxiv2018

Diallel Analysis for Morphological and Biochemical Traits in Tomato Cultivated Under the Influence of Tomato Leaf Curl Virus

Eloquent information about the genetic basis of inheritance is important for any breeding program. Therefore, a diallel study was conducted under the influence of tomato leaf curl virus (TLCV) disease, using the eleven advanced lines of tomato. Firstly, the information regarding percent disease index (PDI) was determined via artificial screening with viruliferous whiteflies. Later, these lines were crossed in half diallel mating design to produce fifty-five one-way hybrids. These hybrids and parental genotypes were evaluated for seven morphological and three biochemical traits under open field conditions. Using the Griffing approach (Method II and Model I) basis of inheritance of traits were determined. Also, a Bayesian model was applied to the total yield descriptor. Correlations data indicated that total yield was not correlated with any other trait. The significant general combining ability (GCA) and specific combining ability (SCA) values indicates exploitable genetic variation. The broad-sense heritability values were larger than narrow-sense heritability, showing that selection will be efficient for the improvement of these traits. Hybrid combinations H23, H42 and H49 can be considered efficient for the selection of multiple traits, including yield. Overall, this study provides a useful information regarding the genetics of important traits of tomato under TLCV infestation.

plant biology↗

bioRxiv2018

Nocturnal expression of Crassulacean acid metabolism (CAM) genes predates the origin of CAM in the genus Yucca.

HighlightAlthough large differences in metabolism exist between C3 and CAM species, we find that many CAM genes have shared expression patterns regardless of photosynthetic pathway, suggesting ancestral propensity for CAM.\n\nAbstractCrassulacean acid metabolism (CAM) is a carbon-concentrating mechanism that has evolved numerous times across flowering plants and is thought to be an adaptation to water limited environments. CAM has been investigated from physiological and biochemical perspectives, but little is known about how plants evolve from C3 to CAM at the genetic or metabolic level. Here we take a comparative approach in analyzing time-course data of C3, CAM, and C3+CAM intermediate Yucca (Asparagaceae) species. RNA samples were collected over a 24-hour period from both well-watered and drought-stressed plants and were clustered based on time-dependent expression patterns. Metabolomic data reveals differences in carbohydrate metabolism and antioxidant response between the CAM and C3 species, suggesting changes to metabolic pathways are important for CAM evolution and function. However, all three species share expression profiles of canonical CAM pathway genes, regardless of photosynthetic pathway. Despite differences in transcript and metabolite profiles between the C3 and CAM species, shared time-structured expression of CAM genes in both CAM and C3 Yucca species suggests ancestral expression patterns required for CAM may have predated its origin in Yucca.

plant biology↗

bioRxiv2018

Alternative splicing and translation play important roles in parallel with transcriptional regulation during rice hypoxic germination

Post-transcriptional mechanisms, including alternative splicing (AS) and alternative translation initiation (ATI), have been used to explain the protein diversity involved in plant developmental processes and stress responses. Rice germination under hypoxia conditions is a classical model system for the study of low oxygen stress. It is known that there is transcriptional regulation during rice hypoxic germination, but the potential roles of AS and ATI in this process are not well understood. In this study, a proteogenomic approach was used to integrate the data from RNA sequencing, qualitative and quantitative proteomics to discover new players or pathways in the response to hypoxia stress. The improved analytical pipeline of proteogenomics led to the identification of 10,253 intron-containing genes, 1,729 of which were not present in the current annotation. Approximately 1,741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds in comparison to controls. Over 95% of these were not present in the list of differentially expressed genes (DEG). In particular, regulatory pathways such as spliceosome, ribosome, ER protein processing and export, proteasome, phagosome, oxidative phosphorylation and mRNA surveillance showed substantial AS changes under hypoxia, suggesting that AS responses are largely independent of traditional transcriptional regulation. Massive AS changes were identified, including the preference usage of certain non-conventional splice sites and enrichment of splicing factors in the DAS datasets. In addition, using self-constructed protein libraries by 6-frame translation, thousands of novel proteins/peptides contributed by ATI were identified. In summary, these results provide deeper insights towards understanding the underlying mechanisms of AS and ATI during rice hypoxic germination.

plant biology↗

bioRxiv2018

Maize defective kernel5 is a bacterial tamB homolog required for chloroplast envelope biogenesis

Chloroplasts are of prokaryotic origin with a double membrane envelope that separates plastid metabolism from the cytosol. Envelope membrane proteins integrate the chloroplast with the cell, but the biogenesis of the envelope membrane remains elusive. We show that the maize defective kernel5 (dek5) locus is critical for plastid membrane biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. We show that dek5 encodes a protein homologous to rice SUBSTANDARD STARCH GRAIN4 (SSG4) and E.coli tamB. TamB functions in bacterial outer membrane biogenesis. The DEK5 protein is localized to the chloroplast envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Both proteomics and antibody-based analyses show that dek5 chloroplasts have reduced levels of chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable metabolite transport.

plant biology↗

bioRxiv2018

Response to early drought stress and identification of QTLs controlling biomass production under drought in pearl millet

Pearl millet plays a major role in food security in arid and semi-arid areas of Africa and India. However, it lags behind the other cereal crops in terms of genetic improvement. The recent sequencing of its genome opens the way to the use of modern genomic tools for breeding. Our study aimed at identifying genetic components involved in early drought stress tolerance as a first step toward the development of improved pearl millet varieties or hybrids. A panel of 188 inbred lines from West Africa was phenotyped under early drought stress and well-irrigated conditions. We found a strong impact of drought stress on yield components. This impact was variable between inbred lines. We then performed an association analysis with a total of 392,493 SNPs identified using Genotyping-by-Sequencing (GBS). Correcting for genetic relatedness, genome wide association study identified QTLs for biomass production in early drought stress conditions and for stay-green trait. In particular, genes involved in the sirohaem and wax biosynthesis pathways were found to co-locate with association loci. Our results open the way to use genomic selection to breed pearl millet lines with improved yield under drought stress.

plant biology↗

bioRxiv2018

Re-annotated Nicotiana benthamiana gene models for enhanced proteomics and reverse genetics

Nicotiana benthamiana is an important model organism and representative of the Solanaceae (Nightshade) family. N. benthamiana has a complex ancient allopolyploid genome with 19 chromosomes, and an estimated genome size of 3.1Gb. Several draft assemblies of the N. benthamiana genome have been generated, however, many of the gene-models in these draft assemblies appear incorrect. Here we present a nearly non-redundant database of 42,855 improved N. benthamiana gene-models. With an estimated 97.6% completeness, the new predicted proteome is more complete than the previous proteomes. We show that the database is more sensitive and accurate in proteomics applications, while maintaining a reasonable low gene number. As a proof-of-concept we use this proteome to compare the leaf extracellular (apoplastic) proteome to a total extract of leaves. Several gene families are more abundant in the apoplast. For one of these apoplastic protein families, the subtilases, we present a phylogenetic analysis illustrating the utility of this database. Besides proteome annotation, this database will aid the research community with improved target gene selection for genome editing and off-target prediction for gene silencing.

plant biology↗

bioRxiv2018

Convergent evolution of effector protease recognition by Arabidopsis and barley

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We show also that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Resistance 1 (Pbr1). Transient co-expression of PBR1 with wild-type AvrPphB, but not a protease inactive mutant, triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 co-immunoprecipitated with barley and N. benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain a Pbr1 ortholog. Phylogenetic analyses showed however that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently, and imply that selection to guard PBS1-like proteins is ancient. Also, the results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

plant biology↗

bioRxiv2018

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley cultivation in Africa and Europe. In contrast to wheat with 82 designated stem rust (Sr) resistance genes, barleys genetic variation for stem rust resistance is very narrow with only seven resistance genes genetically identified. Of these, only one locus consisting of two genes is effective against Ug99, a strain of Pgt which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against Ug99. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

plant biology↗

bioRxiv2018

The molecular signatures of compatible and incompatible pollination

Fertilization in flowering plants depends on the early contact and recognition of pollen grains by the receptive papilla cells of the stigma. To identify the associated molecular pathways, we developed a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. We succeeded in distinguishing 80 % of transcripts according to their parental origins and drew up a catalog of genes whose expression is modified after pollen-stigma interaction. Global analysis of our data reveals that pattern-triggered immunity (PTI)-associated transcripts are upregulated after compatible pollination. From our analysis, we predicted the activation of the Mitogen-activated Protein Kinase 3 on the female side after compatible pollination, which we confirmed through expression and mutant analysis. Our work defines the molecular signatures of compatible and incompatible pollination, highlights the active status of incompatible stigmas, and unravels a new MPK3-dependent cell wall feature associated with stigma-pollen interaction.

plant biology↗

bioRxiv2018

The role of UV-B light on small RNA activity during grapevine berry development

UV-B regulation of anthocyanin biosynthesis in vegetative and grapevine berry tissues has been extensively described. However, its relation with UV-B-regulated microRNAs (miRNAs) has not been addressed before in this species. We explored by deep sequencing of small RNA libraries the developmental dynamics and UV-B effects on miRNAs and associated phased small interfering RNA (phasi-RNAs)-producing loci abundances in in vitro-grown plantlets, in field-grown berry skins of cv. Cabernet Sauvignon, and low- and high UV-B fluence treatments of greenhouse-grown berries at several time points around veraison. We observed by RNA blotting a differential effect of low-versus high-fluence UV-B on miR828 abundances (an effector of anthocyanins and UV-absorbing polyphenolics) across berry development, and identified other miRNAs that correlated with miR828 dynamics. The functional significance of the observed UV-coordinated miRNA responses to UV was supported by degradome evidences of AGO-programmed slicing of mRNAs. Inverse co-expression of the up-regulated miRNAs miR156, miR482, miR530, and miR828 with cognate target gene expressions in response to high fluence UV-B measured by quantitative real-time PCR. These UV-response relationships were also corroborated by analyzing three published transcriptome datasets (berries subjected to UV-C for 1 hr [at pre-veraison], UV-B for five weeks post-veraison, and five red-skinned varieties across four berry development time points). Based on observed significant changes by UV-B on miRNA and derivative phasi-RNA abundances, we propose a regulatory network model of UV responses impacting anti-oxidant and stress-associated polyphenolic compound biosynthesis. In this model high-fluence UV-B increases miR168 (validated in a UV-B small RNA-derived degradome library to target ARGONAUTE1, which spawns phasi-RNAs) and miR530 (targets a novel Plus-3 domain mRNA), while decreasing miR403 abundances (validated to target ARGONAUTE2), thereby coordinating post-transcriptional gene silencing activities by different AGOs. Up-regulation of miR3627/4376 (validated to target Ca2+-transporting ATPase10 that spawns phasi-RNAs) could facilitate anthocyanin accumulation. miR395 and miR399, induced by sulfur and phosphorus starvation in other species (conditions known to trigger anthocyanin accumulation) respond positively to UV-B radiation and are shown to slice cognate targets in grapevine. miR156/miR535 is shown to target SQUAMOSA PROMOTER-BINDING transcription factor genes that potentially regulate the activities of MYB-bHLH-WD40 complexes and thereby anthocyanin biosynthesis. Increases in MYB-bHLH-WD40 TFs could also contribute to the observed up-regulation of miR828 via the conserved and degradome-validated auto-regulatory loop involving miR828/TAS4abc to regulate MYBA6/A7/A5-MYB113-like levels and thereby anthocyanin levels. These results and meta-analysis provide a basis for systems approaches to better understand non-coding RNA functions in response to UV.

plant biology↗

bioRxiv2018

Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of Mediterranean climates, is strongly stimulated by KAR1, which is the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR2. The /{beta}-hydrolase KARRIKIN INSENSITIVE2 (KAI2) is the putative karrikin receptor identified in Arabidopsis. Here we show that B. tournefortii differentially expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR1 relative to KAR2 when expressed in Arabidopsis. We further identify two variant amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer upon weedy ephemerals and potentially other angiosperms differential responses to chemical cues produced by environmental disturbance, including fire.

plant biology↗

bioRxiv2018

PIF transcription factors link a neighbor threat cue to accelerated reproduction in Arabidopsis

Changes in light quality indicative of competition for this essential resource influence plant growth and developmental transitions. Little is known about neighbor proximity-induced acceleration of reproduction. phytochrome B (phyB) senses light cues from plant competitors ultimately leading to the expression of the floral inducers FLOWERING LOCUS (FT) and TWIN SISTER of FT (TSF). Here we show that three PHYTOCHROME INTERACTING FACTOR (PIF) transcriptional regulators act directly downstream of phyB to promote expression of FT and TSF. Neighbor proximity enhances PIF accumulation towards the end of the day coinciding with enhanced floral inducer expression. We present evidence for direct PIF-mediated TSF expression. The relevance of our findings is illustrated by the prior identification of FT, TSF and PIF4 as loci underlying flowering time regulation in nature.\n\nOne Sentence SummaryPIF transcription factors mediate reproductive transition in response to neighbor proximity light cues in Arabidopsis.

plant biology↗

bioRxiv2018

Salinity shock in Jatropha curcas leaves is more pronounced during recovery than during stress time

To verify the possible morphological and ultrastructural differences in the Jatropha curcas leaves, in response to high-intensity salt stress, three genotypes were evaluated (CNPAE183, JCAL171 and CNPAE218). In all the genotypes, 750mM NaCl, added to the nutrient solution, was applied to test its salt tolerance. For the analysis, the leaves were collected at three time points: (i) before stress (time 0 hour); (ii) during stress time (time 50 hours); and (iii) in the recovery period (time 914 hours) when the stressed plants recovered and demonstrated measurements of net photosynthetic with values similar to those demonstrated by the control plants. We showed that regardless of the genotype, saline shock caused an increase in the thickness of the mesophyll, and after the removal of NaCl, the thicker mesophyll remained in the JCAL171 and CNPAE218 genotypes, while the values observed in the CNPAE183 genotype were similar to those before stress. Scanning electron microscopy indicated that the stomata of CNPAE183 are smaller and have a stomatal index higher than the values demonstrated in JCAL171 and CNPAE218. Therefore, among the genotypes analysed, CNPAE183 demonstrates that it could be considered a promising genotype for future studies of genetic improvement that seek elite genotypes tolerant to salinity.\n\nHighlightsThis manuscript present the following highlights:\n\nThe mesophyll thickness contributes to provide a smaller path for the CO2 to Rubisco J. curcas may reduce mesophyll air spaces as a strategy to mitigate low gas exchange Leaves modulate the expansion of stomata differently than other epidermal cells Smaller stomata with greater pore aperture are more abundant on the abaxial surface CNPAE183 is a candidate for studies in search of elite genotypes tolerant to salinity

plant biology↗

bioRxiv2018

Environment-driven genomic and trait divergence in a neotropical mangrove highlight differential sensitivities to climate change

Local adaptation is often a product of environmental variations in the geographical space and has implications for biodiversity conservation. We investigated the role of latitudinal heterogeneity in climate on the organization of genetic and phenotypic variation in the dominant coastal tree, Avicennia schaueriana. In a common garden experiment, samples from an equatorial region, marked by rainy/dry seasons, accumulated less biomass, showed lower stomatal conductance and transpiration, narrower xylem vessels, smaller leaves and higher reflectance of long wavelengths (red light) on the stem epidermis, than samples from a subtropical region, marked by warm/cold seasons. Transcriptome differences identified between trees sampled under field conditions at equatorial and subtropical sites, were enriched in functional categories as responses to temperature, solar radiation, water deficit, photosynthesis and cell wall biosynthesis. The diversity based on thousands of SNP loci revealed a north-south genetic structure. Remarkably, signatures of selection were identified in loci associated with photosynthesis, anthocyanin accumulation and the responses to osmotic and hypoxia stresses. Our results suggest the existence of divergence in key resource-use characteristics, likely driven by climate seasonality, based on water-deficit and solar radiation. These findings provide a basis for conservation plans and for predictions for coastal plant responses to climate change.

plant biology↗

bioRxiv2018

A novel maize gene, glossy6 involved in epicuticular wax deposition and drought tolerance

Epicuticular waxes, long-chain hydrocarbon compounds, form the outermost layer of plant surfaces in most terrestrial plants. The presence of epicuticular waxes protects plants from water loss and other environmental stresses. Cloning and characterization of genes involved in the regulation, biosynthesis, and extracellular transport of epicuticular waxes on to the surface of epidermal cells have revealed the molecular basis of epicuticular wax accumulation. However, intracellular trafficking of synthesized waxes to the plasma membrane for cellular secretion is poorly understood. Here, we characterized a maize glossy (gl6) mutant that exhibited decreased epicuticular wax load, increased cuticle permeability, and reduced seedling drought tolerance relative to wild type. We combined an RNA-sequencing based mapping approach (BSR-Seq) and chromosome walking to identify the gl6 candidate gene, which was confirmed via the analysis of multiple independent mutant alleles. The gl6 gene represents a novel maize glossy gene containing a conserved, but uncharacterized domain. Functional characterization suggests that the GL6 protein may be involved in the intracellular trafficking of epicuticular waxes, opening a door to elucidating the poorly understood process by which epicuticular wax is transported from its site of biosynthesis to the plasma membrane.\n\nSIGNIFICANCE STATEMENTPlant surface waxes provide an essential protective barrier for terrestrial plants. Understanding the composition and physiological functions of surface waxes, as well as the molecular basis underlying wax accumulation on plant surfaces provides opportunities for the genetic optimization of this protective layer. Genetic studies have identified genes involved in wax biosynthesis, extracellular transport, as well as spatial and temporal regulation of wax accumulation. In this study, a maize mutant, gl6 was characterized that exhibited reduced wax load on plant surfaces, increased water losses, and reduced seedling drought tolerance compared to wild type controls. The gl6 gene is a novel gene harboring a conserved domain with an unknown function. Quantification and microscopic observation of wax accumulation as well as subcellular localization of the GL6 protein provided evidence that gl6 may be involved in the intracellular trafficking of waxes, opening a door for studying this necessary yet poorly understood process for wax loading on plant surfaces.

plant biology↗

bioRxiv2018

Molecular dissection of early defense signaling underlying volatile-mediated defense priming and herbivore resistance in rice

Herbivore-induced plant volatiles prime plant defenses and resistance. How volatiles are integrated into early defense signaling is not well understood. Furthermore, whether there is a causal relationship between volatile defense priming and herbivore resistance is unclear. Here, we investigated the impact of indole, a common herbivore-induced plant volatile and known defense priming cue, on early defense signaling and herbivore resistance in rice. We show that rice plants infested by Spodoptera frugiperda caterpillars release up to 25 ng*h-1. Exposure to equal doses of synthetic indole enhances rice resistance to S. frugiperda. Screening of early signaling components reveals that indole directly enhances the expression of the receptor like kinase OsLRR-RLK1. Furthermore, indole specifically primes the transcription, accumulation and activation of the mitogen-activated protein kinase OsMPK3 as well as the expression of the downstream WRKY transcription factor OsWRKY70 and several jasmonate biosynthesis genes, resulting in a higher accumulation of jasmonic acid (JA). Using transgenic plants defective in early signaling, we show that OsMPK3 is required, and that OsMPK6 and OsWRKY70 contribute to indole-mediated defense priming of JA-dependent herbivore resistance. We conclude that volatiles can increase herbivore resistance of plants by priming early defense signaling components.

plant biology↗

bioRxiv2018

Biotrophic interactions disentangled: In-situ localisation of mRNAs to decipher plant and algal pathogen - host interactions at single cell level.

Plant-pathogen interactions follow spatiotemporal developmental dynamics where gene expression in pathogen and host undergo crucial changes. It is of great interest to detect, quantify and localise where and when key genes are active or inactive. Here, we adapt single molecule FISH techniques to demonstrate presence and activity of mRNAs using phytomyxids in their plant and algal host from laboratory and field materials. This allowed to monitor and quantify the expression of genes from the clubroot pathogen Plasmodiophora brassicae, several species of its Brassica hosts, and of several brown algae, including the genome model Ectocarpus siliculosus, infected with the phytomyxid Maullinia ectocarpii. We show that mRNAs are localised along a spatiotemporal gradient, thus providing proof-of-concept of the usefulness of these methods. These methods are easily adaptable to any interaction between microbes and their algal or plant host, and have the potential to increase our understanding of processes underpinning complex plant-microbe interactions.

plant biology↗