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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Gibberellin response in the embryo epidermis regulates germination uniformity in response to seed priming

Uniformity in seed germination remains a primary objective in plant-based food production systems, ensuring predictable and synchronized harvest dates, while suppressing weeds. Treatments including priming can be used to increase germination uniformity and increase the value of commercial seeds. Despite the economic and agronomic importance of seed enhancement treatments, little is known as to how they work at a mechanistic level. Using a combination of molecular genetics and microscopy, we established that hydropriming limits embryo growth genetic programs at an early stage of germination. Conversely, gibberellin (GA) and abscisic acid (ABA)-associated molecular processes progress to later stages of this developmental chronology. The response to GA was specifically affected in the epidermis of germinating embryos in response to hydropriming based on reporter gene expression. The reduction of GA response specifically in the embryo epidermis resulted in increased uniformity of seed germination following hydropriming relative to control seeds. This represents the identification of both a molecular signalling pathway and cell type that are acting to enhance the agronomic germination properties of seed populations. This provides molecular and cellular targets which may be genetically manipulated to enhance seed germination and food production in agronomic species.

plant biology↗

bioRxiv2018

Methyl viologen can affect mitochondrial function in Arabidopsis.

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. The chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in Arabidopsis thaliana, we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies implicate mitochondria in the response to ROS induced by MV.

plant biology↗

bioRxiv2018

Tomato fruit ripening factor NOR controls leaf senescence

NAC transcription factors (TFs) are important regulators of expressional reprogramming during plant development, stress responses and leaf senescence. NAC TFs also play important roles in fruit ripening. In tomato (Solanum lycopersicum), one of the best characterized NAC involved in fruit ripening is NON-RIPENING (NOR) and the non-ripening (nor) mutation has been widely used to extend fruit shelf life in elite varieties. Here, we show that NOR additionally controls leaf senescence. Expression of NOR increases with leaf age, and developmental as well as dark-induced senescence are delayed in the nor mutant, while overexpression of NOR promotes leaf senescence. Genes associated with chlorophyll degradation as well as senescence-associated genes (SAGs) show reduced and elevated expression, respectively, in nor mutants and NOR overexpressors. Overexpression of NOR also stimulates leaf senescence in Arabidopsis thaliana. In tomato, NOR supports senescence by directly and positively regulating the expression of several senescence-associated genes including, besides others, SlSAG15 and SlSAG113, SlSGR1 and SlYLS4. Finally, we find that another senescence control NAC TF, namely SlNAP2, acts upstream of NOR to regulate its expression. Our data support a model whereby NAC TFs have often been recruited by higher plants for both, the control of leaf senescence and fruit ripening.

plant biology↗

bioRxiv2018

A histidine kinase gene is required for large radius root tip circumnutation and surface exploration in rice

The intricate growth patterns that accompany plant organ elongation have long intrigued biologists 1. Circumnutation refers to the circular or elliptical growth of the tip of a plant organ around a central axis. While the utility of circumnutation for climbing plants is clear, its function in roots is less obvious. Additionally, the genetic requirements for root circumnutation are not known. Here we show that mutations in a gene encoding a histidine kinase abolish large radius root tip circumnutation in rice. Using a gel-based imaging system and a whole genome sequenced mutant population, we identified three different mutant alleles of the gene OsHK1 that exhibit increased seedling root depth. Time-lapse imaging indicated that this phenotype is likely due to a lack of large radius root tip circumnutation in OsHK1 mutants. Treatment of mutant roots with the plant hormone zeatin rescues circumnutation, indicating that OsHK1 functions in a cytokinin-related signaling pathway. We found that OsHK1 mutants are impaired in their ability to explore flat surfaces, suggesting that circumnutation facilitates root exploration at the interface of compacted soil horizons.

plant biology↗

bioRxiv2018

FT/FD-GRF5 repression loop directs growth to increase soybean yield

Major advances in crop yield are eternally needed to cope with population growth. To balance vegetative and reproductive growth plays an important role in agricultural yield. To extend vegetative phase can increase crop yield, however, this strategy risks loss of yield in the field as crops may not mature in time before winter come. Here, we identified a repression feedback loop between GmFTL/GmFDL and GmGRF5-1 (Glycine-max-Flowering-Locus-T/Glycine-max-FDL and Glycine-max-GROWTH-REGULATING-FACTOR5-1), which functions as a pivotal regulator in balancing vegetative and reproductive phases in soybean. GmFTL/GmFDL and GmGRF5-1 directly repress gene expression each other. Additionally, GmGRF5-1 enhances vegetative growth by directly enhancing expression of photosynthesis- and auxin synthesis-related genes. To modulate the loop, such as fine-tuning GmFTL expression to trade-off vegetative and reproductive growth, increases substantially soybean yield in the field. Our findings not only uncover the mechanism balancing vegetative and reproductive growth, but open a new window to improve crop yield.

plant biology↗

bioRxiv2018

Thermal acclimation of photosynthetic activity and Rubisco content in two hybrid poplar clones

The mechanistic bases of thermal acclimation of net photosynthetic rate (An) are still difficult to discern and empirical research remains limited, particularly for hybrid poplar. In the present study, we examined the contribution of a number of biochemical and biophysical traits on thermal acclimation of An for two hybrid poplar clones. We grew cuttings of Populus maximowiczii x Populus nigra (MxN) and Populus maximowiczii x Populus balsamifera (MxB) clones under two day/night temperature of 23{degrees}C/18{degrees}C and 33{degrees}C /27{degrees}C and under low and high soil nitrogen level. After 10 weeks, we measured leaf RuBisCO and RuBisCO activase (RCA) amounts and the temperature response of An, dark respiration (Rd), stomatal conductance, (gs), maximum carboxylation rate of CO2 (Vcmax) and photosynthetic electron transport rate (J). Results showed that a 10{degrees}C increase in growth temperature resulted in a shift in thermal optimum (Topt) of An of 6.2{+/-}1.6 {degrees}C and 8.0{+/-}1.2 {degrees}C for clone MxB and MxN respectively, and an increased An and gs at the growth temperature for clone MxB but not MxN. RuBisCO amount was increased by N level but was insensitive to growth temperature while RCA amount and the ratio of its short to long isoform was stimulated by warm condition for clone MxN and at low N for clone MxB. The activation energy of Vcmax and J decreased under warm condition for clone MxB and remain unchanged for clone MxN. Our study demonstrated the involvement of both RCA, activation energy of Vcmax and stomatal conductance in thermal acclimation of An.

plant biology↗

bioRxiv2018

Heterotrimeric G-proteins in unfolded protein response mediate plant growth-defense tradeoffs upstream of steroid and immune signaling

FLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

plant biology↗

bioRxiv2018

SmbHLH37 functions antagonistically with SmMYC2 in regulating jasmonate-mediated biosynthesis of phenolic acids in Salvia miltiorrhiza

Jasmonates (JAs) are integral to various defense responses and induce biosynthesis of many secondary metabolites. MYC2, a basic helix-loop-helix (bHLH) transcription factor (TF), acts as a transcriptional activator of JA signaling. MYC2 is repressed by the JASMONATE ZIM-domain (JAZ) proteins in the absence of JA, but de-repressed by the protein complex SCFCOI1 on perception of JA. We previously reported that overexpression of SmMYC2 promotes the production of salvianolic acid B (Sal B) in Salvia miltiorrhiza. However, the responsible molecular mechanism is unclear. Here, we showed that SmMYC2 binds to and activates the promoters of its target genes SmTAT1, SmPAL1, and SmCYP98A14 to activate Sal B accumulations. SmbHLH37, a novel bHLH gene significantly up-regulated by constitutive expression of SmMYC2, was isolated from S. miltiorrhiza for detailed functional characterization. SmbHLH37 forms a homodimer and interacts with SmJAZ3/8. Overexpression of SmbHLH37 substantially decreased yields of Sal B. SmbHLH37 binds to the promoters of its target genes SmTAT1 and SmPAL1 and blocks their expression to suppress the pathway for Sal B biosynthesis. These results indicate that SmbHLH37 negatively regulates JA signaling and functions antagonistically with SmMYC2 in regulating Sal B biosynthesis in S. miltiorrhiza.

plant biology↗

bioRxiv2018

Cell type-specific differences in protein complex stoichiometry and transcriptional regulation affect the timing of stem cell division

Stem cells divide and differentiate to form all the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the Quiescent Center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here, we gained insight into the differences between the QC and the Cortex Endodermis Initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an Ordinary Differential Equation (ODE) model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction coupled with experimental validation showed that high levels of the SHR-SCR complex is associated with more CEI division but less QC division. Further, our model prediction allowed us to establish the timing of QC and CEI division and propose that SHR repression of QC division depends on the formation of SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.

plant biology↗

bioRxiv2018

Expanding the bioluminescence reporter toolkit for plant chronobiology with NanoLUC

Bioluminescence has been an important tool for gathering circadian data with the main reporter gene exploited firefly luciferase (LUC). In some circumstances the rapid inactivation of LUC could be disadvantageous, e.g. reporting total protein levels through reporter translational fusions. In the latter scenario the commercially available Nano luciferase (NanoLUC) might offer and advantage, however no data in plant has been provided so far. We tested NanoLUC under different research scenarios were LUC has been used, for example enzyme purification, expression in transient plant systems and in stable transgenic lines. We show that NanoLUC is active in these experimental scenarios. We also created a set of NanoLUC variants for example MBP-NanoLUC-3xFlag-10xHis version of NanoLUC can be easily purified and stable for several days (half-life 37.2 at 4{degrees}C) and can be used for generating calibration curves for quantifying protein as the signal is linear over a large dynamic range. In addition, we show that NanoLUC can report in-planta protein levels on circadian time scale thanks to the stability of furimazine. Therefore, opening the possibility of using NanoLUC for reporting protein dynamics in seedlings. With this new technology, we explored the dynamics of protein BROTHER OF LUX ARRHYTHMO (BOA), which has been suggested in mathematical models to be a rhythmic protein from RNA data. Using an automatic plate-reader, we were able to track BOAp:BOA-NL for an extended period of time by just adding a mix of furimazine with Triton X-100, as it is done with LUC-reporter lines. In our experimental context BOA protein does not present strong oscillatory dynamics similar to what has been reported for Phytocrhome B for which rhythmic accumulation of transcript can be observed while total protein levels remain constant under diurnal conditions. Our results support the use NanoLUC for studying the dynamics of plant proteins for extended period of time under different circumstances.

plant biology↗

bioRxiv2018

Positive feedback and feedforward loops between PERIANTHIA, WUSCHEL-RELATED HOMEOBOX5 and GRF-INTERACTING FACTOR 1 modulate gene expression and function in the Arabidopsis root

The Arabidopsis root meristem consists of populations of stem cells that surround the mitotically less active cells known as the Quiescent Center (QC). The QC maintains the stem cells in a non-cell-autonomous manner through the function of the transcription factor (TF) WUSCHEL-RELATED HOMEOBOX5 (WOX5), which is required for columella stem cell (CSC) maintenance. However, whether WOX5 has a regulatory role in any other adjacent stem cells is less understood. To this end, we identified a set of TFs downstream of WOX5 in both QC and Cortex Endodermis Initial (CEI) cells. We then utilized Gene Regulatory Network (GRN) inference to identify GRF-INTERACTING FACTOR 1 (GIF1) as a key gene involved in positive feedback and feedforward loops with WOX5 as well as another stem cell regulator, PERIANTHIA (PAN). Finally, we constructed an ordinary differential equation model based on this inferred GRN to simulate GIF1, PAN, and WOX5 expression over time, which suggests the precise temporal expression of WOX5 and GIF1 is important to sustain QC function.

plant biology↗

bioRxiv2018

OsJAZ9 overexpression improves potassium deficiency tolerance in rice by modulating jasmonic acid levels and signaling

Potassium (K) which makes around 2-10% of plants total dry biomass, when become deficient, makes the plants highly susceptible to both abiotic and biotic stresses. Recent evidences suggest overlapping transcriptional responses to K deficiency and Jasmonate (JA) treatment in plants. However, a link between these responses was missing. Notably, K deficiency and JA application produce similar phenotypic and transcriptional responses. Here, we used molecular, physiological and morphological studies to analyze the role of OsJAZ9 in JA homeostasis, K deficiency and sheath blight resistance. We raised OsJAZ9 overexpression, knockdown, translational reporter and C-terminal deleted translational reporter lines in rice to establish the role of JA signaling in K ion homeostasis and OsJAZ9 as a critical component of JA signaling for K deficiency response. OsJAZ9 overexpression and knockdown provide K deficiency tolerance and sensitivity, respectively, by modulating various K transporters and root system architecture. Furthermore, RNA Seq and JA profiling revealed an elevation of JA responsive genes and JA levels in OsJAZ9 OE lines under K deficiency. Our data provide clear evidence on the crucial role of JAZ repressor, OsJAZ9 in improving K deficiency tolerance in rice by altering JA levels and signaling.

plant biology↗

bioRxiv2018

Solving the riddle of the evolution of Shine-Dalgarno based translation in chloroplasts

The chloroplast, a photosynthetic organelle found in all plant and algae species, originates from an ancient event in which a cyanobacterium was engulfed by a larger eukaryote. Thus, modern chloroplasts still harbor a bacterial-like genome and carry out all stages of gene expression, including mRNA translation by a 70S ribosome. However, the Shine-Dalgarno model, which predominantly regulates translation initiation by base-pairing between the ribosomal RNA and the mRNA in model bacteria genera, was reported to have ambiguous effects on chloroplast gene expression. Here we show that while the Shine-Dalgarno motif is clearly conserved in proteobacterial mRNAs, its general absence from chloroplast mRNAs is observed in cyanobacteria as well, promoting the idea that the evolutionary process of reducing the centrality of the Shine-Dalgarno mechanism began well before plastid endosymbiosis. As plastid ribosomal RNA anti-Shine-Dalgarno elements are highly similar to their bacterial counterparts, these sites alone cannot explain the decline in plastid Shine-Dalgarno generality. However, by computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine-Dalgarno locus, which in-turn reduce the probability of ribosome binding via the Shine-Dalgarno mechanism. To validate our model, we expressed a mCherry gene harboring a Shine-Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. We show that co-expressing it with a 16S ribosomal RNA, modified according to our model, significantly enhances its expression compared to co-expression with an endogenous 16S gene.\n\nSignificance statementChloroplasts are fascinating intracellular organelles which have evolved from an ancient cyanobacterium engulfed by a larger eukaryote. Surprisingly, the canonical mechanism regulating bacterial translation initiation - Shine-Dalgarno - has been shown to play a reduced role in chloroplasts. Here, we show that mutations upstream from the anti-Shine-Dalgarno element decrease the probability of spontaneous ribosome binding by modulating the secondary structure of the ribosomal RNA. These mutations constitute a regulatory step which acclimates the Shine-Dalgarno mechanism to the translational regulation regime of chloroplasts. Interestingly, we show that these chloroplast features occur in modern cyanobacteria as well, promoting the idea that they have evolved prior to endosymbiosis.

plant biology↗

bioRxiv2018

Root volatiles in plant-plant interactions I: Characterization of root sesquiterpene emissions from Centaurea stoebe and their effects on other plants

Volatile organic compounds (VOCs) emitted by plant leaves can influence the physiology of neighboring plants. In contrast to interactions above ground, little is known about the role of VOCs in belowground plant-plant interactions. Here, we characterize constitutive root volatile emissions of the spotted knapweed (Centaurea stoebe) and explore the impact of these volatiles on the germination and growth of different sympatric plant species. We show that C. stoebe roots emit high amounts of sesquiterpenes, with estimated release rates of (E)-{beta}-caryophyllene above 3 g g-1 dw h-1. Sesquiterpene emissions show little variation between different C. stoebe populations, but vary substantially between different Centaurea species. Through root transcriptome sequencing, we identify six root-expressed sesquiterpene synthases (TPSs). Two root-specific TPSs, CsTPS4 and CsTPS5, are sufficient to produce the full blend of emitted root sesquiterpenes. Volatile exposure experiments demonstrate that C. stoebe root volatiles have neutral to positive effects on the germination and growth of different sympatric neighbors. Thus, constitutive root sesquiterpenes produced by two C. stoebe TPSs are associated with facilitation of sympatric neighboring plants. The release of root VOCs may thus influence C. stoebe abundance and plant community structure in nature.

plant biology↗

bioRxiv2018

Root volatiles in plant-plant interactions II: Root terpenes from Centaurea stoebe modify Taraxacum officinale root chemistry and root herbivore growth

Volatile organic compounds (VOCs) emitted by plant roots can influence the germination and growth of neighboring plants. However, little is known about the effects of root VOCs on plant-herbivore interactions. The spotted knapeed (Centaurea stoebe) constitutively releases high amounts of sesquiterpenes into the rhizosphere. Here, we examine the impact of C. stoebe root VOCs on primary and secondary metabolites of sympatric Taraxacum officinale plants and the resulting plant-mediated effects on a generalist root herbivore, the white grub Melolontha melolontha. We show that exposure of T. officinale to C. stoebe root VOCs does not affect the accumulation of defensive secondary metabolites, but modulates carbohydrate and total protein levels in T. officinale roots. Furthermore, VOC exposure increases M. melolontha growth on T. officinale plants. Exposure of T. officinale to a major C. stoebe root VOC, the sesquiterpene (E)-{beta}-caryophyllene, partially mimics the effect of the full root VOC blend on M. melolontha growth. Thus, releasing root VOCs can modify plant-herbivore interactions of neighboring plants. The release of VOCs to increase the susceptibility of other plants may be a form of plant offense.

plant biology↗

bioRxiv2018

Non-functional and weak alleles of FRIGIDA and FLOWERING LOCUS C reduce lifetime water-use independent of leaf-level water-use-efficiency traits in Arabidopsis thaliana

Natural selection driven by water availability has resulted in considerable variation for traits associated with drought tolerance and leaf level water-use efficiency (WUE). In Arabidopsis, little is known about the variation of whole-plant water use (PWU) and whole-plant WUE (TE). To investigate the genetic basis of PWU, we developed a novel proxy trait by combining flowering time and rosette water use to estimate lifetime PWU. We validated its usefulness for large scale screening of mapping populations in a subset of ecotypes. This parameter subsequently facilitated the screening of water-use but also drought tolerance traits in a recombinant inbred line population derived from two Arabidopsis accessions with distinct water use strategies, namely C24 (low PWU) and Col-0 (high PWU). Subsequent quantitative trait loci (QTL) mapping and validation through near-isogenic lines identified two causal QTLs, which showed that a combination of weak and non-functional alleles of the FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) genes substantially reduced plant water-use without penalising reproductive performance. Drought tolerance traits, stomatal conductance, intrinsic water use efficiency ({delta}13C) and rosette water-use were independent of allelic variation at FRI and FLC, suggesting that flowering is critical in determining life-time plant water use, but not leaf-level traits.

plant biology↗

bioRxiv2018

Rhizosphere microbes influence host circadian clock function

The circadian clock is an important determinant of individual fitness that is entrained by local conditions. In addition to known abiotic inputs that entrain the circadian clock, individual pathogenic soil bacteria affect the circadian period of plant hosts. Yet, in nature, plants interact with diverse microbial communities including hundreds to thousands of microbial taxa, and the effect of these communities on clock function remains unclear. In Arabidopsis thaliana, we used diverse rhizosphere inoculates and both wild-type and clock mutant genotypes to test the effect of complex rhizosphere microbial communities on the host circadian clock. Host plants with an intact rhizosphere microbiome expressed a circadian period that was closer to 24 hrs in duration and significantly shorter (by 60 minutes on average) relative to plants grown with a disrupted microbiome. Wild-type host genotypes differed significantly in clock sensitivity to microbiome treatments, where the effect was most pronounced in the Landsberg erecta genotype and least in the Columbia genotype. Rhizosphere microbes collected from a host genotype with a short-period phenotype (toc1-21) and used as inoculate significantly shortened the long-period phenotype of the ztl-1 clock mutant genotype. The results indicate that complex rhizosphere microbial communities significantly affect host clock function.

plant biology↗

bioRxiv2018

Extreme resistance to Potato Virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor

Potato virus Y (PVY) is a major potato pathogen that causes annual losses of billions of dollars. Control of its transmission requires extensive use of environmentally damaging insecticides. Rysto confers extreme resistance (ER) to PVY and is a valuable trait in resistance breeding programs. We isolated Rysto using Resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences Single-Molecule Real Time Sequencing). Rysto encodes a nucleotide binding-leucine rich repeat (NLR) protein with an N-terminal TIR domain, and is sufficient for PVY perception and extreme resistance in transgenic potato plants. We investigated the requirements for Rysto-dependent extreme resistance, and showed that Rysto function is temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.

plant biology↗