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bioRxiv2017

The CLAVATA receptor FASCIATED EAR2 responds to different CLE peptides by signaling through different downstream effectors.

Meristems contain groups of indeterminate stem cells that are critical for organ initiation throughout plant development. The shoot apical meristem (SAM) maintains itself and initiates all shoot organs, such as leaves, floral organs and axillary branch meristems. Development and balanced proliferation of the SAM is regulated by a feedback loop between CLAVATA (CLV) and WUSCHEL (WUS) signaling. CLV signaling is initiated by secretion of the CLV3 peptide ligand, which is perceived directly or indirectly by a number of Leucine-Rich-Repeat (LRR) receptor-like kinases, including CLV1 and BARELY ANY MERISTEM (BAM) 1-3, and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), as well as the receptor-like protein CLV2 in a complex with the CORYNE (CRN) pseudokinase. However, CLV2, and its maize ortholog FASCIATED EAR2 (FEA2) appear to function in signaling by several related CLV3/EMBRYO-SURROUNDING REGION (CLE) peptide ligands, including CLV3. Nevertheless, it remains unknown how CLV2 or FEA2 transmit specific signals from distinct CLE peptides. Here we show that FEA2 is involved in signaling from at least 2 distinct CLE peptides, ZmCLE7, a maize CLV3 ortholog, and ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1), a newly identified CLE peptide functioning in SAM regulation. Signaling from these 2 different CLE peptides appears to be transmitted through 2 different candidate downstream effectors, COMPACT PLANT2 (CT2), the alpha subunit of the maize heterotrimeric G protein, and maize CRN. Our data provide a novel framework to understand how diverse signaling peptides can activate different downstream pathways through common receptor proteins.

plant biology↗

bioRxiv2017

Green microalga Trebouxia sp. produces strigolactone-related compounds

Different algal species that may have germination inducing activity of holoparasitic broomrape weeds Phelipanche aegyptiaca and P. ramosa seeds were screened through germination bioassay. Green alga produce SL-related compounds. Applied extracts of biomass obtained from the culture of green alga Trebouxia arboricola increased seeds germination of both parasites. An optimatization of the alga extraction led to an increase of P. aegyptiaca germination. Exhausted medium also contained SL-related compounds. The crude extract stimulated the roots length of Arabidopsis thaliana tested in vitro. A similar effect had the algae and GR24 aplications on expression levels of the SL-related genes in Physcomitrella patens. The novel analytical method DESI-MSI detected production of carlactone in the algae. The Trebouxia sp. culture applications in pot experiments had positive effect on growth characteristics of pea plants.

plant biology↗

bioRxiv2017

Karrikin-sensing protein KAI2 is a new player in regulating root growth patterns

Roots form highly complex systems varying in growth direction and branching pattern to forage for nutrients efficiently. Here mutations in the KAI2 (KARRIKIN INSENSITIVE) /{beta}-fold hydrolase and the MAX2 (MORE AXILLARY GROWTH 2) F-box leucine-rich protein, which together perceive karrikins (smoke-derived butenolides), caused alteration in root growth direction (root skewing and waving) of Arabidopsis thaliana. This exaggerated root skewing was independent of endogenous strigolactone perception by the D14 /{beta}-fold hydrolase and MAX2. Thus KAI2/MAX2s regulation of root growth may be through perception of endogenous KAI2-ligands, which have yet to be identified. Degradation targets of the KAI2/MAX2 complex, SMAX1 (SUPPRESSOR OF MAX2-1) and SMXL6,7,8 (SUPPRESSOR OF MAX2-1-LIKE) are also involved in the regulation of root skewing. Genetic data reveal a new potential target for degradation, as mutation in the SKS3 (SKU5 similar) but not the SKU5/SKS17 root plasma membrane glycoprotein suppresses the exaggerated root skewing induced by the lack of MAX2. In Arabidopsis thaliana therefore, the KAI2 karrikin-sensing protein acts to limit root skewing, and we propose a mechanism involving root radial expansion as the mutants gravitropic and mechano-sensing responses remained largely unaffected.

plant biology↗

bioRxiv2017

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be easily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

plant biology↗

bioRxiv2017

The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression (Grabowski et al., 2008; Foyer et al., 2014). In this study the putative involvement of WHIRLY1 in stress dependent retrograde signaling was investigated by comparison of barley (Hordeum vulgare L., cv. Golden Promise) wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1. In contrast to the wild type, the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress (Kruszka et al., 2012, Sunkar et al., 2012). The results support a central role of WHIRLY1 in retrograde signaling and underpin a so far underestimated role of microRNAs in this process.

plant biology↗

bioRxiv2017

Scopoletin 8-hydroxylase: a novel enzyme involved in coumarin biosynthesis and iron-deficiency responses in Arabidopsis

HighlightA strongly iron-responsive gene of previously unknown function, At3g12900, encodes a scopoletin 8-hydroxylase involved in coumarin biosynthesis and plays an important role in the iron uptake strategy in Arabidopsis.\n\nAbstractIron (Fe) deficiency represents a serious agricultural problem, particularly in alkaline soils. Secretion of coumarins by Arabidopsis thaliana roots is induced under Fe-deficiency. An essential enzyme for the biosynthesis of major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6-Hydroxylase1 (F6'H1) that belongs to a large enzyme family of the 2-oxoglutarate and Fe(II)-dependent dioxygenases. Another member of this family that is a close homologue of F6H1 and is encoded by a strongly Fe-responsive gene, At3g12900, is functionally characterized in the presented work. We purified the At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin via hydroxylation at the C8-position. Consequently, it was named scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves followed by the metabolite profiling and the biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various regimes of Fe availability. Our results indicate that S8H is involved in coumarin biosynthesis as part of the Fe acquisition machinery.

plant biology↗

bioRxiv2017

Impaired phloem loading in genome-edited triple knock-out mutants of SWEET13 sucrose transporters

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H+ symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. We therefor tested whether SWEETs are important for phloem loading in maize.\n\nHere we identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. Notably, ZmSWEET13 paralogs (a, b, c) are among the highest expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants are severely stunted. Photosynthesis of mutants was impaired and leaves accumulated starch and soluble sugars. RNA-seq revealed profound transcriptional deregulation of genes associated with the photosynthetic apparatus and carbohydrate metabolism. GWAS analyses may indicate that variability in ZmSWEET13s is correlated with agronomical traits, specifically flowering time and leaf angle.\n\nThis work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Zea mays L. Our study highlights these three ZmSWEET13 sucrose transporters as possible candidates for the engineering of crop yield.\n\nOne Sentence Summary: Three SWEET sucrose transporter paralogs are necessary for phloem loading in maize.

plant biology↗

bioRxiv2017

SWEET11 and 15 as key players in seed filling in rice

SummaryO_LIDespite the relevance of seed filling mechanisms for crop yield, we still have only a rudimentary understanding of the pathways and transport processes for supplying the caryopsis with sugars. We hypothesized that the recently identified SWEET sucrose transporters may play important roles in nutrient import pathways in the rice caryopsis.\nC_LIO_LIWe used a combination of mRNA quantification, histochemical analyses, translational promoter-reporter fusions and analysis of knock out mutants created by genomic editing to evaluate the contribution of SWEET transporters to seed filling.\nC_LIO_LIIn rice caryopses, SWEET11 and 15 had the highest mRNA levels and proteins localized to four key sites: the nucellus proper at early stages, the nucellar projection close to the dorsal vein, the nucellar epidermis that surrounds the endosperm, and the aleurone. ossweet11;15 double knock-out lines accumulated starch in the pericarp while caryopses did not contain a functional endosperm.\nC_LIO_LIJointly, SWEET11 and 15 show all hallmarks of being responsible for seed filling with sucrose efflux function at the nucellar projection and transfer across the nucellar epidermis/aleurone interface, delineating two major steps for apoplasmic seed filling, observations that are discussed in relation to observations made in rice and barely on the relative prevalence of these two potential import routes.\nC_LI

plant biology↗

bioRxiv2017

Gene expression dynamics and inter-tissue recognition mechanisms during tissue fusion at the Arabidopsis graft junction

The ability for cut tissues to join together and form a chimeric organism is a remarkable property of many plants, however, grafting is poorly characterized at the molecular level. To better understand this process we monitored genome-wide temporal and spatial gene expression changes in grafted Arabidopsis thaliana hypocotyls. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than the other. This asymmetry correlated with sugar responsive genes and we observed an accumulation of starch above the graft that decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues indicating that a recognition mechanism activated that was independent of functional vascular connections. Auxin which is transported cell-to-cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes were expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, inter-tissue communication and tissue fusion events.

plant biology↗

bioRxiv2017

Using the three dimensional shape of plant cells to predict probabilities of cell division orientation

One key aspect of cell division in multicellular organisms is the orientation of the division plane. Proper division plane establishment contributes to normal organization of the plant body. To determine the importance of cell geometry in division plane orientation, we designed a threedimensional probabilistic mathematical modeling approach to directly test the century-old hypothesis that cell divisions mimic \"soap-film minima\" or that daughter cells have equal volume and the resulting division plane is a local surface area minimum. Predicted division planes were compared to a plant microtubule array that marks the division site, the preprophase band (PPB). PPB location typically matched one of the predicted divisions. Predicted divisions offset from the PPB occurred when a neighboring cell wall or PPB was observed directly adjacent to the predicted division site, to avoid creating a potentially structurally unfavorable four-way junction. By comparing divisions of differently shaped plant and animal cells to divisions simulated in silico, we demonstrate the generality of this model to accurately predict in vivo division. This powerful model can be used to separate the contribution of geometry from mechanical stresses or developmental regulation in predicting division plane orientation.

plant biology↗

bioRxiv2017

NAC-NOR mutations in tomato Penjar accessions attenuate multiple metabolic processes and prolong the fruit shelf life

Several Penjar accessions of tomato (Solanum lycopersicum), widely grown in the Mediterranean region, exhibit prolonged shelf life, and harbor alcobaca mutation with valine-106-aspartic acid substitution in the NAC-NOR protein. To uncover the metabolic basis underlying the prolonged shelf life, we compared four Penjar accessions to Ailsa Craig (AC). Three accessions bore alcobaca mutation, whereas fourth was a novel NAC-NOR allele with only six amino acids in the encoded protein. The cuticle composition among Penjars varied widely during the progression of fruit ripening. All Penjars exhibited delayed ripening, prolonged on-vine and off-vine shelf life, low ethylene emission and carotenoid levels albeit with accession-specific differences. Metabolic profiling revealed shifts in Krebs cycle intermediates, amino acids, and {beta}-aminobutyric acid levels indicating the attenuation of respiration in Penjars during post-harvest storage. The prolonged shelf life of Penjar fruits was associated with a concerted downregulation of a number of cell-wall modifying genes and cell-wall-related metabolites. The accumulation of higher ABA and sucrose levels at the onset of senescence in Penjar fruits likely contribute to reduced water loss. Our analyses reveal that in addition to specialized cuticle composition, the attenuation of various metabolic processes by NAC-NOR mutation likely prolongs the shelf life of Penjar fruits.\n\nHighlightThe prolonged shelf life of tomato Penjar accessions bearing mutations in NAC-NOR transcription factor appears to be regulated by a combined effect of attenuation of respiration, altered cuticle composition, enhanced ABA and sucrose levels in fruits and downregulation of cell wall modification

plant biology↗

bioRxiv2017

Nitrate modulates stem cell dynamics by regulating WUSCHEL expression through cytokinins

The shoot apical meristem (SAM) is responsible for the generation of all of the aerial parts of plants1. Given its critical role, dynamical changes in SAM activity should play a central role in the adaptation of plant architecture to the environment2. Using quantitative microscopy, grafting experiments and genetic perturbations, we connect the plant environment to the SAM, by describing the molecular mechanism by which cytokinins signal the level of nutrient availability to the SAM. We show that a systemic signal of cytokinin precursors3 mediates the adaptation of SAM size and organogenesis rate to the availability of mineral nutrients by modulating the expression of WUSCHEL, a key regulator of stem cell homeostasis4. In time-lapse experiments, we further show that this mechanism allows meristems to adapt to rapid changes in nitrate concentration, and thereby modulate their rate of organ production to the availability of mineral nutrients within a few days. Our work sheds new light on the role of the stem cell regulatory network, by showing that it does not only maintain meristem homeostasis but also allows plants to adapt to rapid changes in the environment.

plant biology↗

bioRxiv2017

Characterization of Thylakoid Division Using Chloroplast Dividing Mutants in Arabidopsis

Chloroplasts are double membrane bound organelles that are found in plants and algae. Their division requires a number of proteins to assemble into rings along the center of the organelle and to constrict in synchrony. Chloroplasts possess a third membrane system, the thylakoids, which house the majority of proteins responsible for the light-dependent reactions. The mechanism that allows chloroplasts to sort out and separate the intricate thylakoid membrane structures during organelle division remain unknown. By characterizing the sizes of thylakoids found in a number of different chloroplast division mutants in Arabidopsis, we show that thylakoids do not divide independently of the chloroplast division cycle. More specifically, we show that thylakoid division requires the formation of both the inner and the outer contractile rings of the chloroplast.

plant biology↗

bioRxiv2017

Measurement of leaf day respiration using a new isotopic disequilibrium method compared with the Laisk method

O_LIQuantification of leaf respiration is of great importance for the understanding of plant physiology and ecosystem biogeochemical processes. Leaf respiration continues in light (RL) but supposedly at a lower rate compared to the dark (RD). Yet, there is no method for direct measurement of RL and most available methods require unphysiological measurement conditions.\nC_LIO_LIA method based on isotopic disequilibrium quantified RL (RL 13C) and mesophyll conductance of young and old fully-expanded leaves of six species compared RL 13C to RL values determined by the Laisk method (RL Laisk).\nC_LIO_LIRL 13C and RL Laisk were consistently lower than RD. Leaf ageing negatively affected photosynthetic performance, but had no significant effect on RL or RL/RD as determined by both methods. RL Laisk and RL 13C were measured successively on the same leaves and correlated positively (r2=0.38), but average RL Laisk was 28% lower than RL13C. Using A/Cc curves instead of A/Ci curves, a higher photocompensation point {Gamma}* (by 5 mol mol-1) was found but the correction had no influence on RL Laisk estimates.\nC_LIO_LIThe results suggest that the Laisk method underestimated RL. The isotopic disequilibrium method is useful for assessing responses of RL to irradiance and CO2, improving our mechanistic understanding of RL.\nC_LI

plant biology↗

bioRxiv2017

Identification and characterization of Gypsophila paniculata color morphs in Sleeping Bear Dunes National Lakeshore, MI, USA.

BackgroundGypsophila paniculata (babys breath) is an invasive species found throughout much of the northwest United States and western Canada. Recently, plants exhibiting a different color morphology were identified within the coastal dunes along eastern Lake Michigan. The common babys breath (G. paniculata) typically produces stems that are purple in color (purple morph), while the atypical morph has stems that are green-yellow (green-yellow morph). The purpose of this study was to characterize these newly identified morphs and determine if they are genetically distinct species from the common babys breath in order to assess whether alternative management strategies should be employed to control these populations.\n\nMethodsWe sequenced two chloroplast regions, rbcL and matK, and one nuclear region, ITS2, from the purple morphs and green-yellow morphs collected from Sleeping Bear Dunes National Lakeshore, MI, USA (SBDNL). Sequences were aligned to the reference sequences from other Gypsophila species obtained from the Barcode of Life (BOLD) and GenBank databases. We also collected seeds from wild purple morph and wild green-yellow morph plants in SBDNL. We grew the seeds in a common garden setting and characterized the proportion of green-yellow individuals produced from the two color morphs after five-months of growth.\n\nResultsPhylogenetic analyses based upon rbcL, matK, and ITS2 regions suggest that the two color morphs are not distinct species and they both belong to G. paniculata. Seeds collected from wild green-yellow morphs produced a significantly higher proportion of green-yellow individuals compared to the number produced by seeds collected from wild purple morphs. However, seeds collected from both color morphs produced more purple morphs than green-yellow morphs.\n\nDiscussionBased upon these results, we propose that the two color morphs are variants of G. paniculata. Given the significant difference in the number of green-yellow morphs produced from the seeds of each morph type, we also suggest that this color difference has some genetic basis. We propose that current management continue to treat the two color morphs in a similar manner in terms of removal to prevent the further spread of this species.

plant biology↗

bioRxiv2017

Leaf rolling in maize crops: from leaf scoring to canopy level measurements for phenotyping

Leaf rolling in maize crops is one of the main plant reactions to water stress that may be visually scored in the field. However, the leaf scoring did not reach the high-throughput desired by breeders for efficient phenotyping. This study investigates the relationship between leaf rolling score and the induced canopy structure changes that may be accessed by high-throughput remote sensing techniques.\n\nResults gathered over a field phenotyping platform run in 2015 and 2016 show that leaf starts to roll for the water stressed conditions around 9:00 and reaches its maximum around 15:00. Conversely, genotypes conducted under well watered conditions do not show any significant rolling during the same day. Leaf level rolling was very strongly correlated to canopy structure changes as described by the fraction of intercepted radiation fIPARWS derived from digital hemispherical photography. The changes in fIPARWS were stronly correlated (R2=0.86, n=50) to the leaf level rolling visual score. Further, a very good consistency of the genotype ranking of the fIPARWS changes during the day was found ({rho}=0.62). This study demonstrating the strong coordination between leaf level rolling and its impact on canopy structure changes poses the basis for new high-throughput remote sensing methods to quantify this water stress trait.\n\nHighlighThe diurnal dynamics of leaf rolling scored visually is strongly related to canopy structure changes that can be documented using Digital hemispherical photography. Consequences for high-throughput field phenotyping are discussed

plant biology↗

bioRxiv2017

Sexual lineage specific DNA methylation regulates Arabidopsis meiosis

DNA methylation controls eukaryotic gene expression and is extensively reprogrammed to regulate animal development. However, whether developmental methylation reprogramming during the sporophytic life cycle of flowering plants regulates genes is presently unknown. Here we report a distinctive, gene-targeted RNA-directed DNA methylation (RdDM) activity in the Arabidopsis thaliana male sexual lineage that regulates gene expression in meiocytes. Loss of sexual lineage-specific RdDM causes mis-splicing of the MPS1/PRD2 gene, thereby disrupting meiosis. Our results establish a regulatory paradigm in which de novo methylation creates a cell-lineage-specific epigenetic signature that controls gene expression and contributes to cellular function in flowering plants.

plant biology↗

bioRxiv2017

What’s in the box? Authentication of Echinacea herbal products using DNA metabarcoding and HPTLC

BackgroundDifferences in regulatory policies between countries as well as a lack of appropriate standardized methods for authentication and quality control of herbal products lead to concerns over quality and safety. Echinacea products are among the top selling herbal products in Europe and the United States with indications for broad range of ailments.\n\nPurposeThis study approached the need for a novel analytical strategy in authentication of herbal products.\n\nMethodsA combination of high performance thin layer chromatography (HPTLC) and DNA metabarcoding was employed. Fifty-three Echinacea herbal products marketed across Europe were tested to evaluate the accuracy of these methods in plant identification and their potential in detecting substitutes, adulterants and other unreported plant constituents.\n\nResultsHPTLC provides a high resolution in detecting Echinacea phytochemical target compounds, but does not offer information on the other species within the product. Alternatively, we showed that the limitation of HPTLC to detect non-targeted species can be overcome through the complementary use of DNA metabarcoding. Using DNA metabarcoding, Echinacea species were detected in 34 out of the 38 retained products (89 %), but with a lack of discriminatory resolution at the species level due to the low level of molecular divergence within the Echinacea genus. All herbal products showed considerable discrepancies between ingredients listed on the label and the ones detected using DNA metabarcoding registering an overall ingredient fidelity of 43 %.\n\nConclusionThe results confirm that DNA metabarcoding can be used to test for the presence of Echinacea and simultaneously to detect other species present in even highly processed and multi-ingredient herbal products.\n\nAbbreviations

plant biology↗