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bioRxiv2017

VlbZIP30 of grapevine functions in drought tolerance via the abscisic acid core signaling pathway

Drought stress limits the growth and development of grapevines, thereby reducing productivity, but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized. Here, we characterized a group A bZIP gene from Kyoho grapevine, VlbZIP30, which was shown to be induced by abscisic acid (ABA) and dehydration stress. Overexpression of VlbZIP30 in transgenic Arabidopsis enhanced dehydration tolerance during seed germination, and in the seedling and adult stages. Transcriptome analysis revealed that a major proportion of ABA- and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage. We identified an A. thaliana G-box motif (CACGTG) and a potential grapevine G-box motif (MCACGTGK) in the promoters of the 39 selected A. thaliana genes up-regulated in the transgenic plants and in the 35 grapevine homologs, respectively. Subsequently, using two grapevine-related databases, we found that 74% and 84% (a total of 27 genes) of the detected grapevine genes were significantly up-regulated by ABA and drought stress, respectively, suggesting that these 27 genes involve in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine. We propose that VlbZIP30 functions as a positive regulator of drought-responsive signaling in the ABA core signaling pathway.\n\nHighlightVlbZIP30 positively regulate plant drought tolerance through regulated the expression of 27 grapevine candidate genes via G-box cis-element (MCACGTGK) in ABA signaling pathway.

plant biology↗

bioRxiv2017

A Genetic Pathway Composed of EDT1/HDG11, ERECTA, and E2Fa Loci Regulates Water Use Efficiency by Modulating Stomatal Density

Improvement of crop drought resistance and water use efficiency (WUE) has been a major endeavor in agriculture. ERECTA is the first identified major effector of water use efficiency. However, the underlying molecular mechanism is not well understood. Here, we report a genetic pathway, composed of EDT1/HDG11, ERECTA, and E2Fa loci, which regulates water use efficiency by modulating stomatal density. The HD-START transcription factor EDT1/HDG11 transcriptionally activates ERECTA expression by binding to an HD cis-element in the ERECTA promoter. ERECTA in turn relies on E2Fa to control the expression of cell-cycle related genes and the transition from mitosis to endocycle, which leads to increased nuclear DNA content in leaf cells, and therefore increased cell size and decreased stomatal density. The decreased stomatal density improves plant WUE. Our study demonstrates the EDT1/HDG11-ERECTA-E2Fa genetic pathway that reduces stomatal density by increasing cell size, providing a new avenue to improve WUE of crops.

plant biology↗

bioRxiv2017

An epigenetic breeding system in soybean for increased yield and stability

Epigenetic variation has been associated with a wide range of adaptive phenotypes in plants, but there exist few direct means for exploiting this variation. RNAi suppression of the plant-specific gene, MutS HOMOLOG1 (MSH1), in multiple plant species produces a range of developmental changes accompanied by modulation of defense, phytohormone, and abiotic stress response pathways. This msh1-conditioned developmental reprogramming is retained independent of transgene segregation, giving rise to transgene-null memory effects. An isogenic memory line crossed to wild type produces progeny families displaying increased variation in adaptive traits that respond to selection. This study investigates amenability of the MSH1 system for inducing epigenetic variation in soybean that may be of value agronomically. We developed epi-line populations by crossing with msh1-acquired soybean memory lines. Derived soybean epi-lines showed increase in variance for multiple yield-related traits including pods per plant, seed weight, and maturity time in both greenhouse and field trials. Selected epi-F2:4 and epi-F2:5 lines showed an increase in seed yield over wild type. By epi-F2:6, we observed a return of MSH1-derived enhanced growth back to wild type levels. Epi-populations also showed evidence of reduced epitype-by-environment (e x E) interaction, indicating higher yield stability. Transcript profiling of the soybean epi-lines identified putative signatures of enhanced growth behavior across generations. Genes related to cell cycle, abscisic acid biosynthesis, and auxin-response, particularly SMALL AUXIN UP RNAs (SAURs), were differentially expressed in epi-F2:4 lines that showed increased yield when compared to epi-F2:6. These data support the potential of msh1-derived epigenetic variation in plant breeding for enhanced yield and yield stability.

plant biology↗

bioRxiv2017

Exploring the Genetics of Lesion and Nodal Resistance in Pea (Pisum sativum L.) to Sclerotinia sclerotiorum Using Genome-wide Association Studies and RNA-Seq

The disease white mold caused by the fungus Sclerotinia sclerotiorum is a significant threat to pea production and improved resistance to this disease is needed. Nodal resistance in plants is a phenomenon where a fungal infection is prevented from passing through a node and the infection is limited to an internode region. Nodal resistance has been observed in some pathosystems such as the pea (Pisum sativum L.)-S. sclerotiorum pathosystem. Other than nodal resistance, different pea lines display different levels of stem lesion size restriction, referred to as lesion resistance. It is unclear whether the genetics of lesion resistance and nodal resistance are identical or different. This study applied genome-wide association studies (GWAS) and RNA-Seq to understand the genetic makeup of these two types of resistance. The time series RNA-Seq experiment consisted of two pea lines (the susceptible Lifter and the partially resistant PI 240515), two treatments (mock samples and S. sclerotiorum inoculated samples), and three time points (12, 24, and 48 hours post-inoculation). Integrated results from GWAS and RNA-Seq analyses identified different redox-related transcripts for lesion and nodal resistances. A transcript encoding a glutathione S-transferase was the only shared resistance source for both phenotypes. There were more leucine rich-repeat containing transcripts found for lesion resistance, while different candidate resistance transcripts such as a VQ motif-containing protein and a myo-inositol oxygenase were found for nodal resistance. This study demonstrated the robustness of combining GWAS and RNA-Seq for identifying white mold resistance in pea, and results suggest different genetics underlying lesion and nodal resistance.

plant biology↗

bioRxiv2017

Bundle-sheath cells are internal leaf "water valves" controlled via xylem acidification by H+-ATPase

O_LIThe leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem-sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem-sap pH of <6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSCs proton pump, AHA2, we now test the hypothesis that it regulates this pH and leaf radial water fluxes. C_LIO_LIWe monitored the xylem-sap pH in the veins of detached leaves of WT Arabidopsis, AHA mutants, and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor and stimulator, and different pH buffers. We monitored their impact on the xylem-sap pH and the whole leaf hydraulic conductance (Kleaf), and the effect of pH on the water osmotic permeability (Pf) of isolated BSCs protoplasts. C_LIO_LIOur results demonstrated that AHA2 is necessary for xylem-sap acidification, and in turn, for elevating Kleaf. Conversely, knocking out AHA2 alkalinized the xylem-sap. Also, elevating xylem sap pH to 7.5 reduced Kleaf and elevating external pH to 7.5 decreased the BSCs Pf. C_LIO_LIAll these demonstrate a causative link between AHA2 activity in BSCs and leaf radial water conductance. C_LI One-sentence summaryBundle-sheath cells can control the leaf hydraulic conductance by proton-pump-regulated xylem sap pH

plant biology↗

bioRxiv2017

Trait components of whole plant water use efficiency are defined by unique, environmentally responsive genetic signatures in the model C4 grass Setaria

Plant growth and water use are interrelated processes influenced by the genetic control of both plant morphological and biochemical characteristics. Improving plant water use efficiency (WUE) to sustain growth in different environments is an important breeding objective that can improve crop yields and enhance agricultural sustainability. However, genetic improvements of WUE using traditional methods have proven difficult due to low throughput and environmental heterogeneity encountered in field settings. To overcome these limitations the study presented here utilizes a high-throughput phenotyping platform to quantify plant size and water use of an interspecific Setaria italica x Setaria viridis recombinant inbred line population at daily intervals in both well-watered and water-limited conditions. Our findings indicate that measurements of plant size and water use in this system are strongly correlated; therefore, a linear modeling approach was used to partition this relationship into predicted values of plant size given water use and deviations from this relationship at the genotype level. The resulting traits describing plant size, water use and WUE were all heritable and responsive to soil water availability, allowing for a genetic dissection of the components of plant WUE under different watering treatments. Linkage mapping identified major loci underlying two different pleiotropic components of WUE. This study indicates that alleles controlling WUE derived from both wild and domesticated accessions of the model C4 species Setaria can be utilized to predictably modulate trait values given a specified precipitation regime.

plant biology↗

bioRxiv2018

Mapping and dynamics of regulatory DNA during seed development

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of pectin. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation, enriching for seed coat cells with the INTACT method. We found over 3000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with seven-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, in this comparison, development exerted a stronger effect on the plant regulatory landscape than extreme environmental perturbations, highlighting the importance of extending studies of regulatory landscapes to other tissues and cell types during development.

plant biology↗

bioRxiv2017

Multiple strategies for heat adaptation in rice endosperms revealed by on-site cell-specific analysis

Plant cells have multiple strategies to adapt to environmental stresses. Rice endosperms form chalkiness in a part of the tissue under heat conditions during the grain-filling stage, although nitrogen supply reduces chalky rice. Air spaces formed in the cells cause an irregular light reflection and create chalkiness, yet what exactly occurs remains unclear at cell level. Through on-site cell-specific analysis, we show that heat-treated cells adjust osmotically and retard protein synthesis to preserve protein storage vacuoles in the cytosol, resulting in air space formation. Application of nitrogen enhances heat tolerance to sustain protein body and amyloplast development during strong osmotic adjustment, which diminishes air spaces to avoid chalkiness. Hence, we conclude that rice endosperm cells could alter organelle compartments spatially during the heat adaptation, depending on the available nitrogen level. Our findings provide new insight into the cellular mechanism of rice chalky formation as a strategy for heat acclimation.

plant biology↗

bioRxiv2017

Blue Light Negatively Regulates Tolerance to Phosphate Deficiency in Arabidopsis

Plants have evolved mechanisms to improve utilization efficiency or acquisition of inorganic phosphate (Pi) in response to Pi deficiency, such as altering root architecture, secreting acid phosphatases, and activating the expression of genes related to Pi uptake and recycling. Although many genes responsive to Pi starvation have been identified, transcription factors that affect tolerance to Pi deficiency have not been well characterized. We show here that defect in the ELONGATED HYPOCOTYL 5 (HY5) transcription factor gene results in tolerance to Pi deficiency in Arabidopsis. The primary root length of hy5 was only slightly inhibited under Pi deficient condition and its fresh weight was significantly higher than that of wild type. The Pi deficiency-tolerant phenotype of hy5 was similarly observed when grown on the medium without Pi. In addition, a double mutant, hy5slr1, without lateral roots also showed tolerance to phosphate deficiency, indicating that the tolerance of hy5 does not result from increase of external Pi uptake and may be related to internal Pi utilization or recycling. Moreover, we found that blue light negatively regulates tolerance to Pi-deficiency and that hy5 exhibits tolerance to Pi deficiency due to blockage of blue-light responses. Collectively, this study points out light quality may play an important role in the regulation of internal Pi recycling and utilization efficiency. Also, it may contribute to reducing Pi fertilizer requirements in plants through a proper illumination.

plant biology↗

bioRxiv2017

Fluorescent reporter lines for auxin and cytokinin signalling in barley (Hordeum vulgare)

The phytohormones auxin and cytokinin influence the development and maintenance of plant stem cell niches. Although barley (Hordeum vulgare) is the fourth most abundant cereal crop plant, the knowledge about these important phytohormones in regard to the root and shoot stem cell niche in barley is still negligible. In this study, we analyse the influence of auxin and cytokinin on the barley root meristem and present reporter lines to describe the auxin and cytokinin signalling output. Application of high concentrations of auxin and cytokinin to barley seedlings had a negative influence on barley root and meristem growth. The expression of the cytokinin reporter TCSn revealed that cytokinin signalling mostly takes place in the stele cells proximal to the QC and in the differentiated root cap cells, but can additionally be activated in the root stem cell niche by cytokinin application. Analysing signalling targets of auxin showed that a homologue of AtPLT1, HvPLT1, is expressed in a similar way as AtPLT1 in Arabidopsis, in particular in the QC and the surrounding cells. Furthermore, a homologue of the auxin PIN transporters PIN1, HvPIN1, was expressed in the root and the shoot meristem and polarly localizes to the plasma membrane. Its expression is regulated by cytokinin and the intracellular localisation is affected by BFA. With this study, we provide a valuable tool set of fluorescent barley reporter lines for auxin and cytokinin.

plant biology↗

bioRxiv2017

Synthetic hormone-responsive transcription factors can monitor and re-program plant development

Developmental programs continuously sculpt plant morphology to meet environmental challenges, and these same programs have been manipulated to increase agricultural productivity1,2. Small molecule phytohormones act as signals within these programs creating chemical circuitry3 that, in many cases, has been represented in mathematical models4,5. To date, model validation and forward engineering of plant morphology has been largely restricted to adding or subtracting genes, as more nuanced tools to modulate key control parameters identified by such models in vivo are severely limited6,7. Here, we use Arabidopsis thaliana to validate a novel set of synthetic and modular hormone activated Cas9-based repressors (HACRs) that respond to three phytohormones: auxin, gibberellins and jasmonates. We demonstrate that HACRs can regulate genes in response to both exogenous hormone treatments, as well as in response to local differences in endogenous hormone levels associated with developmental events. We further show that HACRs can be used to reprogram the agriculturally relevant traits of shoot branching and phyllotaxy by tuning canalization strength, a critical control parameter predicted by mathematical models. By deploying a HACR to re-parameterize the threshold for induction of the auxin transporter PIN-FORMED1 (PIN1), we observed a decrease in shoot branching and phyllotactic noise as predicted by existing models4,5. The approach described here provides a framework for improved mapping of developmental circuitry, as well as a means to better leverage model predictions to engineer development.

plant biology↗

bioRxiv2017

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

plant biology↗

bioRxiv2017

The co-regulatory interaction of transpiration and water potential: Implications for stomatal conductance models and height growth in trees

Leaf water potential decreases with increasing transpiration rate according to an analogue of Ohms law, while transpiration rate decreases with decreasing leaf water potential in the framework of stomatal control. This interaction is not accommodated in present-day models of stomatal conductance. We formally derive the equilibrium between these two counteracting processes for steady-state water conditions. We show that the mechanism considered causes an attenuation of the immediate effect of atmospheric variables on transpiration, which can improve existing models of stomatal conductance that presume noninterdependent variables. Parameters from European beech (Fagus sylvatica L.) are used to illustrate the results.

plant biology↗

bioRxiv2017

A model of protein interactions for regulating plant stem cells

The plant shoot apical meristem holds a stem cell niche from which all aerial organs originate. Using a computational approach we show that a mixture of monomers and heterodimers of the transcription factors WUSCHEL and HAIRY MERISTEM is sufficient to pattern the stem cell niche, and predict that immobile heterodimers form a regulatory pocket surrounding the stem cells. The model achieves to reproduce an array of perturbations, including mutants and tissue size modifications. We also show its ability to reproduce the recently observed dynamical shift of the stem cell niche during the development of an axillary meristem. The work integrates recent experimental results to answer the longstanding question of how the asymmetry of expression between the stem cell marker CLAVATA3 and its activator WUSCHEL is achieved, and recent findings of plasticity in the system.

plant biology↗

bioRxiv2017

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals novel siRNA loci

Plant small RNAs regulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. sRNAs fall into two major categories: those that are reliant on RNA Dependent RNA Polymerases (RDRs) for biogenesis and those that arent. Known RDR-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR-independent sRNAs are primarily microRNAs and other hairpin-derived sRNAs. In this study, we produced and analyzed small RNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. Only a small fraction of all sRNA loci were RDR1/RDR2/RDR6-independent; most of these were microRNA loci or associated with predicted hairpin precursors. We found 58 previously annotated microRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent small RNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for small RNA biogenesis. These 38 small RNA-producing loci have novel biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggest that these 38 loci represent one or more new types of small RNAs in Arabidopsis thaliana.\n\nSignificance StatementSmall RNAs regulate gene expression in plants and are produced through a variety of previously-described mechanisms. Here, we examine a set of previously undiscovered small RNA-producing loci that are produced by novel mechanisms.

plant biology↗

bioRxiv2017

Comparative analysis of genetic diversity and differentiation of cauliflower (Brassica oleracea var. botrytis) accessions from two ex situ genebanks

Cauliflower (Brassica oleracea var. botrytis) is an important vegetable crop for human nutrition. We characterized 192 cauliflower accessions from the USDA and IPK genebanks with genotyping by sequencing (GBS). They originated from 26 different countries and represent about 44% of all cauliflower accessions in both genebanks. The analysis of genetic diversity revealed that accessions formed two major groups that represented the two genebanks and were not related to the country of origin. This differentiation was robust with respect to the analysis methods that included principal component analysis, ADMIXTURE and neighbor-joining trees. Genetic diversity was higher in the USDA collection and significant phenotypic differences between the two genebanks were found in three out of six traits investigated. GBS data have a high proportion of missing data, but we observed that the exclusion of single nucleotide polymorphisms (SNPs) with missing data or the imputation of missing SNP alleles produced very similar results. The results indicate that the composition and type of accessions have a strong effect on the structure of genetic diversity of ex situ collections, although regeneration procedures and local adaptation to regeneration conditions may also contribute to a divergence. Fst-based outlier tests of genetic differentiation identified only a small proportion (<1%) of SNPs that are highly differentiated between the two genebanks, which indicates that selection during seed regeneration is not a major cause of differentiation between genebanks. Seed regeneration procedures of both genebanks do not result in different levels of genetic drift and loss of genetic variation. We therefore conclude that the composition and type of accessions mainly influence the level of genetic diversity and explain the strong genetic differentiation between the two ex situ collections. In summary, GBS is a useful method for characterizing genetic diversity in cauliflower genebank material and our results suggest that it may be useful to incorporate routine genotyping into accession management and seed regeneration to monitor the diversity present in ex situ collections and to reduce the loss of genetic diversity during seed regeneration.

plant biology↗

bioRxiv2017

Auxin regulates endosperm cellularization in Arabidopsis

The endosperm is an ephemeral tissue that nourishes the developing embryo, similar to the placenta in mammals. In most angiosperms endosperm development starts as a syncytium, where nuclear divisions are not followed by cytokinesis. The timing of endosperm cellularization largely varies between species and the event triggering this transition remains unknown. Here we show that increased auxin biosynthesis in the endosperm prevents its cellularization, leading to seed arrest. Auxin-overproducing seeds phenocopy paternal-excess triploid seeds derived from hybridizations of diploid maternal plants with tetraploid fathers. Concurrently, auxin-related genes are strongly overexpressed in triploid seeds, correlating with increased auxin activity. Reducing auxin biosynthesis and signaling reestablishes endosperm cellularization in triploid seeds and restores their viability, highlighting a causal role of increased auxin in preventing endosperm cellularization. We propose that auxin determines the time of endosperm cellularization and thereby uncovered a central role of auxin in establishing hybridization barriers in plants.

plant biology↗

bioRxiv2017

An unconventional NOI/RIN4 domain of a rice NLR protein binds host EXO70 protein to confer fungal immunity

As much as 10% of plant immune receptors from the nucleotide-binding domain leucine-rich repeat (NLR) family carry integrated domains (IDs) that can directly bind pathogen effectors. However, it remains unclear whether direct binding to effectors is a universal feature of ID-containing NLRs given that only a few NLR-IDs have been functionally characterized. Here we show that the rice (Oryza sativa) sensor NLR-ID Pii2 confers resistance to strains of the rice blast fungus Magnaporthe oryzae that carry the effector AVR-Pii without directly binding this protein. First, we show that AVR-Pii binds the exocyst subunit OsExo70F2 in rice (Oryza sativa) to dissociate preformed complexes of OsExo70F2 with host RPM1 INTERACTING PROTEIN4 (RIN4) at the conserved NOI motif, facilitating a possible virulence function. Second, we show that in its resting state, Pii2 binds OsExo70F2 and OsExo70F3, essential components of Pii-mediated resistance, through its integrated NOI domain. Remarkably, AVR-Pii binding to OsExo70F2/F3 leads to dissociation of the Pii2-OsExo70F2 and Pii2-OsExo70F3 complexes, destabilization of Pii2, and activation of immunity. These findings support a novel conceptual model in which an NLR-ID monitors alterations of tethered host proteins targeted by pathogen effectors, providing insight into pathogen recognition mechanisms. Significance statementPlant diseases diminish crop yields by over 20% each year, and deploying resistant crops is the most effective way to combat them. Nucleotide-binding domain leucine-rich repeat (NLR)-type receptors are the major player in plant resistance against pathogens, with a subset of NLRs containing unconventional domains called integrated domains (ID) derived from host proteins. Previous studies suggest that pathogen avirulence (AVR) effectors directly bind or modify NLR-IDs before they are recognized by the host. Here, we reveal that the rice NLR-ID receptor Pii2 indirectly recognizes AVR-Pii when the effector dissociates Pii2 from the host Exo70 proteins tethered to Pii2. We propose a new model of how NLRs can recognize pathogens, expanding our understanding of plant immunity.

plant biology↗