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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2015

Genomic Diversity and Climate Adaptation in Brachypodium

The Brachypodium genus contains the model grasses B. distachyon, B. stacei and B. hybridum, that are useful for molecular and physiological studies relevant to grain, pasture and bioenergy crops, as well as ecology. In this chapter we discuss the natural variation in climate/geography, genotypic and phenotypic diversity that exists within these species. We describe utilisation of this diversity via two methods, Genome Wide Association Studies and Landscape Genomics, to examine the interaction between specific genetic variants, phenotype, and environment. The aim is to identify adaptive loci that control specific traits in specific environments and understand the contribution of background polygenetic variation shaped by demographic processes. With recent developments in high throughput phenotyping, cheaper genotyping by sequencing and higher spatial/temporal resolution of climate data, these approaches can exploit the diversity of the Brachypodium. Experiments using this toolkit will reveal alleles, genes and pathways underlying agriculturally important and environmentally sensitive traits for use in grass breeding.

Plant Biology↗

bioRxiv2015

ExoRNAi exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNAi-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, stp1 and stp2 in tomato seedlings triggers corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponds directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observe no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato, and potentially other crop plants.

Plant Biology↗

bioRxiv2015

Processes driving nocturnal transpiration and implications for estimating land evapotranspiration

Evapotranspiration is a major component of the water cycle, yet only daytime transpiration is currently considered in Earth system and agricultural sciences. This contrasts with physiological studies where 25% or more of water losses have been reported to occur occurring overnight at leaf and plant scales. This gap probably arose from limitations in techniques to measure nocturnal water fluxes at ecosystem scales, a gap we bridge here by using lysimeters under controlled environmental conditions. The magnitude of the nocturnal water losses (12-23% of daytime water losses) in row-crop monocultures of bean (annual herb) and cotton (woody shrub) would be globally an order of magnitude higher than documented responses of global evapotranspiration to climate change (51-98 vs. 7-8 mm yr-1). Contrary to daytime responses and to conventional wisdom, nocturnal transpiration was not affected by previous radiation loads or carbon uptake, and showed a temporal pattern independent of vapour pressure deficit or temperature, because of endogenous controls on stomatal conductance via circadian regulation. Our results have important implications from large-scale ecosystem modelling to crop production: homeostatic water losses justify simple empirical predictive functions, and circadian controls show a fine-tune control that minimizes water loss while potentially increasing posterior carbon uptake.

Plant Biology↗

bioRxiv2015

Developmental and phenotypic plasticity of Arabidopsis thaliana accessions across an ambient temperature range

BackgroundGlobal increase in ambient temperatures constitute a significant challenge to wild and cultivated plant species. Forward genetic analyses of individual temperature-responsive traits have resulted in the identification of several signaling and response components. However, a comprehensive knowledge about temperature sensitivity of different developmental stages and the contribution of natural variation is still scarce and fragmented at best.\n\nResultsHere, we systematically analyze thermomorphogenesis throughout a complete life cycle in ten natural Arabidopsis thaliana accessions grown in four different temperatures ranging from 16 to 28 {degrees}C. We used Q10, GxE, phenotypic divergence and correlation analyses to assess temperature sensitivity and genotype effects of more than 30 morphometric and developmental traits representing five phenotype classes. We found that genotype and temperature differentially affected plant growth and development with variing strengths. Furthermore, overall correlations among phenotypic temperature responses was relatively low which seems to be caused by differential capacities for temperature adaptations of individual accessions.\n\nConclusionGenotype-specific temperature responses may be attractive targets for future forward genetic approaches and accession-specific thermomorphogenesis maps may aid the assessment of functional relevance of known and novel regulatory components.

Plant Biology↗

bioRxiv2015

Latent developmental and evolutionary shapes embedded within the grapevine leaf

O_LIAcross plants, leaves exhibit profound diversity in shape. As a single leaf expands, its shape is in constant flux. Plants may also produce leaves with different shapes at successive nodes. Additionally, leaf shape varies among individuals, populations, and species due to evolutionary processes and environmental influences.\nC_LIO_LIBecause leaf shape can vary in many different ways, theoretically the effects of distinct developmental and evolutionary processes are separable, even within the shape of a single leaf. Here, we measure the shapes of >3,200 leaves representing >270 vines from wild relatives of domesticated grape (Vitis spp.) to determine if leaf shapes attributable to genetics and development are separable from each other.\nC_LIO_LIWe isolate latent shapes (multivariate signatures that vary independently from each other) embedded within the overall shape of leaves. These latent shapes can predict developmental stages independent from species identity and vice versa. Shapes predictive of development are then used to stage leaves from 1,200 varieties of domesticated grape (Vitis vinifera), revealing that changes in timing underlie leaf shape diversity.\nC_LIO_LIOur results indicate distinct latent shapes combine to produce a composite morphology in leaves, and that developmental and evolutionary contributions to shape vary independently from each other.\nC_LI

Plant Biology↗

bioRxiv2015

The Cf-4 receptor-like protein associates with the BAK1 receptor-like kinase to initiate receptor endocytosis and plant immunity

O_LIThe first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the RLK SUPPRESSOR OF BIR1 (SOBIR1) contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. To address this, we investigated functioning of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum.\nC_LIO_LIWe employed live-cell imaging and co-immunoprecipitation in tomato and Nicotiana benthamiana to investigate the requirement of associated kinases for Cf activity and ligand-induced subcellular trafficking of Cf-4.\nC_LIO_LIUpon elicitation with the matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1 (BAK1) associates with Cf-4 and Cf-9. Furthermore, Cf-4 that interacts with SOBIR1 at the plasma membrane, is recruited to late endosomes after elicitation. Significantly, BAK1 is required for Avr4-triggered endocytosis, effector-triggered defenses in Cf-4 plants and resistance of tomato against C. fulvum.\nC_LIO_LIOur observations indicate that RLP-mediated immune signaling and endocytosis require ligand-induced recruitment of BAK1, reminiscent of BAK1 interaction and subcellular fate of the FLAGELLIN SENSING 2 RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for signaling initiation and endocytosis.\nC_LI

Plant Biology↗

bioRxiv2015

Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double strand breaks in barley

Transcription activator-like effector nucleases (TALENs) open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can be fused with effector domains like the nucleolytically active part of FokI in order to induce double strand breaks (DSBs) and thereby modify the host genome on a predefined target site via non-homologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific TALENs along with a repair template that, via HDR, facilitates conversion of gfp into yfp which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected YFP accumulation in about 3 out of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. Predictable genetic modifications comprising only a few genomic base pairs rather than entire genes are of particular practical relevance, because they might not fall under the European regulation of genetically engineered organisms. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.

Plant Biology↗

bioRxiv2015

Expression of E. coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch-like polyglucans

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of (1[->]6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes (DBEs) that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and DBEs is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water-insolubility, crystallinity, presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen branching enzyme (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis that is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs origin only have a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.

Plant Biology↗

bioRxiv2015

Tomato I2 immune receptor can be engineered to confer partial resistance to the oomycete Phytophthora infestans in addition to the fungus Fusarium oxysporum

Plants and animals rely on immune receptors, known as nucleotide-binding domain and leucine-rich repeat containing proteins (NB-LRR or NLR), to defend against invading pathogens and activate immune responses. How NLR receptors respond to pathogens is inadequately understood. We previously reported single-residue mutations that expand the response of the potato immune receptor R3a to AVR3aEM, a stealthy effector from the late blight oomycete pathogen Phytophthora infestans. I2, another NLR that mediates resistance to the wilt causing fungus Fusarium oxysporum f. sp. lycopersici, is the tomato ortholog of R3a. We transferred previously identified R3a mutations to I2 to assess the degree to which the resulting I2 mutants have an altered response. We discovered that wild-type I2 protein responds weakly to AVR3a. One mutant in the N-terminal coiled-coil domain, I2I141N, appeared sensitized and displayed markedly increased response to AVR3a. Remarkably, I2I141N conferred partial resistance to P. infestans. Further, I2I141N has an expanded response spectrum to F. oxysporum f. sp. lycopersici effectors compared to the wild-type I2 protein. Our results suggest that synthetic immune receptors can be engineered to confer resistance to phylogenetically divergent pathogens and indicate that knowledge gathered for one NLR could be exploited to improve NLRs from other plant species.

Plant Biology↗

bioRxiv2015

Phenotypic analysis of Arabidopsis transgenic plants constitutively expressing the P6 protein from Cauliflower mosaic virus or mutant alleles thereof

This laboratory has previously shown that nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4. EMBO J., 27:2102 - 2112. In the original paper, there were inadvertent errors in the mounting of a figure. We have now repeated the experiments concerned, using the seed stock of the corresponding transgenic plants. The results presented here confirm those reported in the original paper. The plants expressing P6 are strongly stunted with chlorotic and serrated leaves. Conversely, the transgenic expression of various P6 mutant alleles do not induce this phenotype.

Plant Biology↗

bioRxiv2015

The P25 protein of Potato virus X suppresses accumulation of 24nt siRNAs in the N. benthamiana transgenic line 16c

By transiently expressing viral suppressors of RNA silencing (VSRs) in combination with GFP silencing-inducing constructs in leaves of Nicotiana benthamiana line 16c, Hamilton et al. (2002) could establish a positive correlation between the production of 24nt GFP small interfering (si)RNAs in infiltrated leaves and the systemic onset of GFP silencing in remote tissues. In the context of GFP silencing inducers based on replicating Potato virus X (PVX), the P25 protein of PVX was found to specifically inhibit the local accumulation of 24nt GFP siRNAs. In the original paper, there were background pixel pattern duplications in the figure reporting the P25 experiments. We have now repeated these experiments with the original clones and the results presented here confirm those reported in the original paper.

Plant Biology↗

bioRxiv2015

Light-induced indeterminacy alters shade avoiding tomato leaf morphology

Plants sense foliar shade of competitors and alter their developmental programs through the shade avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of >18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits, but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light-responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier.\n\nObserving that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia shade avoidance is not mediated through canonical pathways described in mature organs, but rather the expression of KNOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from prolonged production of juvenile leaf types.

Plant Biology↗

bioRxiv2015

Ancient trans-acting siRNAs Confer Robustness and Sensitivity onto the Auxin Response

Novel developmental programs often evolve via cooption of existing genetic networks. To understand this process, we explored cooption of the TAS3 tasiRNA pathway in the moss Physcomitrella patens. We find an ancestral function for this repeatedly redeployed pathway in the spatial regulation of a conserved set of Auxin Response Factors. In moss, this results in stochastic patterning of the filamentous protonemal tissue. Through modeling and experimentation, we demonstrate that tasiRNA regulation confers sensitivity and robustness onto the auxin response. Increased auxin sensitivity parallels increased developmental sensitivity to nitrogen, a key environmental signal. We propose that the properties lent to the auxin response network, along with the ability to stochastically modulate development in response to environmental cues, have contributed to the tasiRNA-ARF modules repeated cooption during evolution. The signaling properties of a genetic network, and not just its developmental output, are thus critical to understanding the evolution of multicellular forms.

Plant Biology↗

bioRxiv2015

Single molecule sequencing of THCA synthase reveals copy number variation in modern drug-type Cannabis sativa L.

O_LICannabinoid expression is an important genetically determined feature of cannabis that presents clinical and legal implications for patients seeking cannabinoid specific therapies like Cannabidiol (CBD).\nC_LIO_LICannabinoid, terpenoid, and flavonoid marker assisted selection can accelerate breeding efforts by offering genetic tools to select for desired traits at an early stage in growth. To this end, multiple models for chemotype inheritance have been described suggesting a complex picture for chemical phenotype determination.\nC_LIO_LIHere we explore the potential role of copy number variation of THCA Synthase using phased single molecule sequencing and demonstrate that copy number and sequence variation of this gene is common and suggests a more nuanced view of chemotype prediction.\nC_LI

Plant Biology↗

bioRxiv2015

Genetic transformation of micropropagated shoots of Pinus radiata D.Don

Key Message Agrobacterium tumefaciens was used to transform radiata pine shoots and to efficiently produce stable genetically modified pine plants.\n\nAbstract Micropropagated shoot explants from Pinus radiata D. Don were used to produce stable transgenic plants by Agrobacterium tumefaciens-mediated transformation. Using this method any genotype that can be micropropagated could produce stable transgenic lines. As over 80% of P. radiata genotypes tested can be micropropagated, this effectively means that any line chosen for superior characteristics could be transformed. There are well-established protocols for progressing such germplasm to field deployment. Here we used open and control pollinated seed lines and embryogenic clones. The method developed was faster than other methods previously developed using mature cotyledons. PCR positive shoots could be obtain within 6 months of Agrobacterium co-cultivation compared with 12 months for cotyledon methods. Transformed shoots were obtained using either kanamycin or geneticin as the selectable marker gene. Shoots were recovered from selection, were tested and were not chimeric, indicating that the selection pressure was optimal for this explant type. GFP was used as a vital marker, and the bar gene, (for resistance to the herbicide Buster(R)) was used to produce lines that could potentially be used in commercial application. As expected, a range of expression phenotypes were identified for both these reporter genes and the analyses for expression were relatively easy.

Plant Biology↗

bioRxiv2015

Reconstitution of CO2 regulation of SLAC1 anion channel and function of CO2-permeable PIP2;1 aquaporin as carbonic anhydrase 4 interactor

Daily dark periods cause an increase in the leaf CO2 concentration (Ci) and the continuing atmospheric [CO2] rise also increases Ci. Elevated Ci causes closing of stomatal pores thus regulating gas exchange of plants. The molecular signaling mechanisms leading to CO2-induced stomatal closure are only partially understood. Here we demonstrate that high intracellular [Formula] enhances currents mediated by the guard cell S-type anion channel SLAC1 when co-expressing either of the protein kinases OST1, CPK6 or CPK23 in Xenopus oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the {beta}CA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation and co-immunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Co-expression of {beta}CA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified which abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity in Xenopus oocytes. These findings identify the CO2-permeable PIP2;1 aquaporin as key interactor of carbonic anhydrases, show functional reconstitution of extracellular CO2 signaling to ion channel regulation and implicate SLAC1 as a bicarbonate-responsive protein in CO2 regulation of S-type anion channels.

Plant Biology↗

bioRxiv2015

Whole genome duplication in coast redwood (Sequoia sempervirens) and its implications for explaining the rarity of polyploidy in conifers

SO_SCPLOWUMMARYC_SCPLOWO_LIWhereas polyploidy is common and an important evolutionary factor in most land plant lineages it is a real rarity in gymnosperms. Coast redwood (Sequoia sempervirens) is the only hexaploid conifer and one of just two naturally polyploid conifer species. Numerous hypotheses about the mechanism of polyploidy in Sequoia and parental genome donors have been proffered over the years, primarily based on morphological and cytological data, but it remains unclear how Sequoia became polyploid and why this lineage overcame an apparent gymnosperm barrier to whole-genome duplication (WGD).\nC_LIO_LIWe sequenced transcriptomes and used phylogenetic inference, Bayesian concordance analysis, and paralog age distributions to resolve relationships among gene copies in hexaploid coast redwood and its close relatives.\nC_LIO_LIOur data show that hexaploidy in the coast redwood lineage is best explained by autopolyploidy or, if there was allopolyploidy, this was restricted to within the Californian redwood clade. We found that duplicate genes have more similar sequences than would be expected given evidence from fossil guard cell size which suggest that polyploidy dates to the Eocene.\nC_LIO_LIConflict between molecular and fossil estimates of WGD can be explained if diploidization occurred very slowly following whole genome duplication. We extrapolate from this to suggest that the rarity of polyploidy in conifers may be due to slow rates of diploidization in this clade.\nC_LI

Plant Biology↗

bioRxiv2015

Climate and developmental plasticity: interannual variability in grapevine leaf morphology

The shape of leaves are dynamic, changing over evolutionary time between species, within a single plant producing different shaped leaves at successive nodes, during the development of a single leaf as it allometrically expands, and in response to the environment. Notably, strong correlations between the dissection and size of leaves with temperature and precipitation exist in both the paleorecord and extant populations. Yet, a morphometric model integrating evolutionary, developmental, and environmental effects on leaf shape is lacking. Here, we continue a morphometric analysis of >5,500 leaves representing 270 grapevines of multiple Vitis species between two growing seasons. Leaves are paired one-to-one, vine-to-vine accounting for developmental context, between growing seasons. Linear Discriminant Analysis reveals shape features that specifically define growing season, regardless of species or developmental context. The shape feature, a more pronounced distal sinus, is associated with the colder, drier growing season, consistent with patterns observed in the paleorecord. We discuss the implications of such plasticity in a long-lived woody perennial, such as grapevine, with respect to the evolution and functionality of plant morphology and changes in climate.

Plant Biology↗