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The monocot grass, Setaria viridis, is an emerging C4 model specie in the field of plant biology and biotechnology. Setaria viridis, is a diploid plant utilizing C4 photosynthesis. It belongs to the Poaceae family, subfamily Panicoideae, which comprises the most agronomical important grass crops such as the cereals maize, sorghum and millet. Members of the Panicoideae subfamily of grasses grow crown roots from their shoots a characteristic that is not observed in many well studied eudicots such as Arabidopsis and Tomato. While the crown roots sustained growth and branching allow optimal soil exploration, the hydraulic conductivity of those roots favors the radial transfer of water from the soil for subsequent transport to the shoot. However, the molecular and physiological mechanisms that regulate crown-root hydraulics are poorly understood. Aquaporins, which are water channel proteins that facilitate water transport across root cell membranes, could be important components of radial crown-root water transport. Although they represent a key point for the adaptation of wild plant species to diverse natural habitats and are a major target for crop improvement under climate change scenarios, little is known about the signaling mechanisms that link soil properties to crown-root hydraulics and aquaporin functions, particularly in grasses. Therefore, we will use two Panicoideae model plants, Setaria viridis accessions that differ in their crown-root traits and stress responses (Saha, Sade et al., 2016) to study the genetic and molecular mechanisms that control crown-root hydraulic conductivity and root-to-shoot gas-exchange signaling under variable environmental conditions. Our proposed study will aim to answer the following questions: What is the contribution of AQP to crown-root hydraulics and how does that contribution compare to the contribution of root morphology (i.e., xylem structure and development). What are the most prominent crown root-specific AQPs? What role (s) do these individual AQPs play in determining radial root hydraulics and the whole-plant gas-exchange rate? To what extent are crown-root AQPs involved in mediating the whole-plant response to drought? What are the genetic components that regulate crown-root hydraulics in grass plants under changing environmental conditions? To address these questions, we will employ the following approaches in crown-root tissue from the two S. viridis accessions: (i) transcriptome analysis to determine the prominent crown-root AQP, (ii) analysis of crown-root cell water permeability (using protoplasts) and AQP activity (using AQP blockers), (iii) anatomical (cross-sectional) and developmental (in situ root imaging) analysis of crown roots, (iv) analysis of crown root to shoot gas-exchange signaling (LI-COR and PlantDitech), (v) crown root-specific overexpression and knockout of an AQP, (vi) genomic and transcriptomic comparison of the two S. viridis accessions and (vii) generation of recombinant inbred lines (RILs) and a genetic map for the identification of SNPs and genes associated with crown-root traits. This project will yield unique information on molecular and genomic changes related to root and whole-plant water use and stress tolerance, and the research findings will be a valuable resource for grass research and improvement of grass crops. This work will highlight the importance of water conductivity in different types of roots and will expand our knowledge of the molecular basis of aquaporin-mediated stress signals, root hydraulics and whole-plant transpiration in grasses. Furthermore, this study will also yield a wealth of new, high-quality data regarding root tissue-specific optimal and stress-related gene-expression profiles for the model plant Setaria viridis.

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Developing  perennials grains for a sustainable agriculture in a changing climate

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Perennial grain cropping systems provide many beneficial attributes for the environment as compared to their annual analogues . Perennial crops are more resource-use efficiency and contribute less to soil erosion. The domestication of the perennial Intermediate Wheatgrass (IWG) for grain and forage production is being developed and a semi-commercial variety is currently used (i.e. Kernza®) in USA and Canada.  However, the effect(s) of domestication on the response(s) to abiotic stress of  Kernza is not understood and the feasibility of expanding the growth of IWG to Mediterranean conditions (i.e. hot summers) has not been tested. The overall objective of this research is to identify physiological and biochemical traits for breeding perennial grain crops able to growth under Mediterranean-like stress conditions. We will compare the responses of domesticated and non-domesticated perennial IWG varieties grown under optimal and abiotic stress conditions to identify traits associated with the stress response and traits that are associated with crop domestication. We will apply a Systems Biology approach, combining physiology, biochemistry, genomics, metabolomics and bioinformatics to identify genes and pathways that will define useful traits to crop breeders. The development of stress-tolerant perennial grain varieties is an important priority in the present scenario of climate change, soil erosion and diminishing food security. The outcome of the research  will greatly contribute to the identification of key traits associated with sustainable growth and enhanced yield of perennial grains, growing under stress conditions that are typical to Mediterranean climates. In addition, key traits identified in the work done on the commercial KERNZA  (e.g., N/C homeostasis, stress induced chloroplast degradation and water relations)  will be functionally characterized using using gain-of-function (gene expression under the control of stress-induced promoters) and loss-of-function (gene silencing and gene editing – CRISPR/Cas 9) approaches in a perennial grass model plant Brachypodium Sylvaticum. Brachypudium sylvaticum is a perennial grass that is a suitable model for the study of perennial grass. We have studied the abiotic stress tolerance of  WT accessions using physiology (whole plant measurements) cell biology (e.g cell and tissue sodium transport), genomics (RNAseq) and biochemistry (metabolomics and lipidomics) and identify suitable genetic material  for the study of perennial crops response to climate change.

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Research questions and scientific approach

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Project 1: Physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas-exchange signaling in Setaria viridis under variable environmental conditions

We wish to understand basic questions such those pertaining to the physiological and metabolic pathways that regulate source‒sink relations and water homeostasis in crop plants (e.g., wheat, Arabidopsis, tomato) under changing climate conditions (e.g., more severe drought, heat, salinity). For example, differences in root morphology, root hydraulics and leaf senescence can all affect modified source‒sink relations. We applying both reverse genetics (CRISPR/CAS9) and forward genetics (genome wide association mapping and correlation network analysis combined with machine learning) approaches on crop plants  (both wild and domesticated) to identify genes and pathways associated with physiological traits important for climate change scenarios.

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Sustained growth and branching of grasses (e.g., the Panicoideae subfamily, which is particularly important for agriculture) roots allows for optimal soil exploration. However,  the water permeability of grasses roots (in other words, root hydraulic conductivity) favors the radial transfer of water from the soil for subsequent transport to the shoot. Nevertheless, the molecular and physiological mechanisms that regulate grasses root hydraulics are a matter of ongoing research. Aquaporins (AQPs; water-channel proteins) facilitate the transport of water across root cell membranes. AQPs could be main effectors of radial root water transport (alongside other as yet unknown regulators). Although they represent a key point for the adaptation of wild plant species to diverse natural habitats and are a major target for crop improvement, the signaling mechanisms that link soil properties with root hydraulics and AQP functions remain largely unknown, especially in grasses. The main goal of this project was to gain better understanding of the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas-exchange signaling (e.g., water use efficiency). The work was conducted in the model cereal plant Setaria viridis under variable environmental conditions. Using physiological (i.e., gas exchange and root hydraulics), molecular (i.e., gene editing, expression analysis), biochemical (i.e., AQP inhibitors, protein‒protein interaction) and cellular (i.e., cell water permeability and cell localization) tools, we have identified a unique AQP which is a  candidate for the regulation of root hydraulics and shoot gas exchange. Our findings provide a novel aspects for the role of AQPs in the regulation of physiological traits in Setaria Viridis.

Project 2: Phenotypical and molecular characterization of the CHLOROPLAST VESICULATION (CV) gene in Solanum lycopersicum – a novel target in fleshy-fruit crops for the improvement of abiotic-stress tolerance via the enhancement of source capacity under drought and saline conditions

Achieving nutritional stability and food security is a major challenge facing agriculture in our times. Dramatic climate changes and the increasing rate of population growth drive us to continually seek to improve yield quality and quantity. One of the main approaches to achive this goal is through the cultivation of abiotic-stress tolerance in crops. Tomato (Solanum lycopersicum), one of the most important non-grain crops, is highly sensitive to climate conditions. Changes in patterns of rainfall, temperature regimes and outlier weather events all affect the productivity of tomato by changing the source‒sink interactions within the plant. This project aims to improve source capacity and stability under drought and saline conditions and to improve yields through the manipulation of a novel target gene – CHLOROPLAST VESICULATION (CV). A set of cellular-localization techniques, gene knockout via CRISPR/cas9, measurements of gene expression (real-time PCR) and several physiological techniques were applied to study the CV gene. We found that tomato CV is localized in the chloroplasts and that, when activated, it contributes to chloroplast degradation. Furthermore, we showed that homozygous CV knockout plants (CVCRISPR) react significantly better to salinity and drought stress, and also exhibit an improved rate of CO2 fixation, higher chlorophyll concentrations enhance source/sink relations (carbon and nitrogen assimilation and allocation). Furthermore, our  homozygous CV knockout lines showed better yield production , quality and harvest index under stress conditions. We perform a deep characterization of sink tissue (fruits) under stress  (i.e., fruit metabolomics and transcriptomic) and used correlation network analysis and machine learning to identify downstream  metabolic pathways and genes that regulate sink capacity and quality under stress-induced senescence conditions in tomato. We identified that the L-glutamine and L-arginine biosynthesis pathways are associated with stress-response conditions and highlight putative novel genes involved in tomato fruit quality under stress. Our findings provide a novel aspects for the role of CV in the regulation of source/sink relations  in tomatoes. Furthermore, genes identified in our transcriptomic data are being validated and expected to provide new insight to the regulation of fruit quality.

Project 3: Regulation of stomata function, ROS formation, water use efficiency and drought tolerance in tomato by ROP GTPase signaling

Increasing global temperatures and declining freshwater resources possess a serious and immediate threat to human and livestock food supply. Therefore, the development of crops with improved water efficiency (WUE) and drought tolerance is one of the most important tasks of agricultural biotechnology. The coupling between CO2 uptake and water transpiration through stomatal pores present an inherent problem for increasing WUE since decreases in stomatal pore size while reducing transpiration often also result in insufficient CO2 uptake, reduction in photosynthetic CO2 assimilation and reduced growth and crop yields. The present research  is based upon findings by Prof Shaul Yalovsky and Dr Nir Sade  Labs that tomato (Solanum lycopersicum) loss-of-function mutants in a small GTP binding protein Rho Of Plants 9 (ROP9) displayed increased WUE and drought tolerance in mid-size greenhouse experiments with preliminary evidence for yield improvement due to modified guard cell signaling that results in increased formation of NADPH oxidase (RBOH)-dependent ROS formation.  We hypothesize that activated ROP9 interacts with and represses RBOH function. Inactivation of ROPs by ABA leads to RBOH activation and ROS formation. ROP loss of function by-passes ABA-dependent ROP inactivation leading to constitutive increase in guard cell ROS and partial stomatal closure. ROP9 and possibly ROP9 signaling components could be targets for increasing WUE without compromising yield in tomato in field conditions. Our research objectives and work plan are: 1. To substantiate and expand the mid-size greenhouse, field and yield analyses. We are carring out a large-scale field phenotyping and yield analysis of the rop9 mutants’ performance under well-watered and several partial drought condition, 2. measure and quantify rop9 mutants’ water use efficiency at the leaf and whole plant using several independent methodologies and measure gas exchange and photosynthesis of rop9 mutants under defined environmental conditions and 3. use stomata specific transcriptomics to examine the link between changes in gene expression and increased ROS in rop9 mutants and identify novel targets for improving WUE. Our overall goals are to Investigate the potential of ROP9 as target for increasing WUE and drought tolerance in field conditions and determine of the underlying molecular mechanisms. Our findings provide a novel aspects for the role of ROPs in the regulation of water use efficiency in tomatoes together with applied aspects of the generation of field grown efficient tomatoes.

Project 4: Exploring root hydraulics and root system architecture (RSA) traits plasticity under stress conditions using genome wide association mapping (GWAS) in diverse genetic population of crop and model plants

Abiotic stress is a key factor of crop yield limitation, particularly water deficit (WD) that has the greatest effect on crop productivity. Water scarcity threatens crop production in dry growing regions in many parts of the world. Changing climatic conditions will likely exacerbate this situation as extreme soil moisture deficits and warmer summers are predicted. To optimize production under these conditions, growers will need plant material that better tolerates abiotic stress. work is needed to elucidate the mechanisms that enable roots to tolerate dry conditions by maintaining soil water acquisition and growth. Revealing plants reaction to those stresses, focusing on plants water use for optimal growth and functioning has become a crucial issue worldwide.
Root system architecture (RSA) is described as the three-dimensional structure of the root system distribution in the soil. Whole-plant water uptake is influenced by RSA and root hydraulic properties. Root depth is a key parameter of water uptake when water is accessible in deeper soil levels, but the investigation of overall root system structure is more complicated due to the plasticity response to heterogeneous distribution of soil resources.
Arabidopsis (model plant) and wheat (crop) represent a good genetic foundation to study the genetic and molecular bases of root traits and adaptive responses. Recent advancement in genetic editing technology, such as CRISPR, provides the tools to create specific mutant plants. Thus, the influence of a single gene on plant’s phenotype can be validated by experiment using mutants.
Genome-wide association studies (GWAS) is a method to identify inherited genetic variants in different individuals associated with a phenotype of interest. This approach scans many individual’s genome to evaluate associations between hundreds of thousands of single-nucleotide polymorphisms (SNPs) and a specific trait. Insight of the genetic architecture can be provided by GWAS, so an inform decision regarding transgenic and mutagenesis candidates can be made. A comparison of two GWAS of different environments, such as control and abiotic stress, can help to reveal which genes influenced stress responses. Thus, GWAS is an exciting methodology that can help us locate the genomic loci, and specific genes, that are linked to root architecture and root hydraulic.
Our main goal is to identify novel genes that effect root response (i.e. root growth and hydraulic conductivity) in various environment. We will focus mainly on root response to various ABA, salinity and water deficient  levels. An untargeted GWAS and statistical analysis is used to reveal candidate genes that might associate with root phenotype.  Up to date, we have perform a high throughput phenotype screening of : 1) 250 accessions of dicot Arabidopsis root system architecture under control and different stress conditions  2) 200 accessions of monocot  Aegilops longissima ( a specie with great scientific interest because it contains a wide range of desired trait and is phylogenetically the most closely related to wheat) root system architecture under control and different stress conditions (salinity  and drought).  We are in the process of analyzing the data and identifying target genes that regulate root system architecture in both dicot and monocot under variable conditions. Finally, custom mutant plants will be made to validate the linkage between the discovered genes and root phenotypes, and to understand their pathway. Furthermore, the candidates genes will be tested for their relations to other root traits such as root hydraulics.  

Project 5: A systems-biology approach to elucidating the metabolic pathways for the abiotic-stress tolerance of wild emmer wheat

One major focus in agriculture today is enhancing crop productivity under stress conditions (e.g., drought, heat and salinity). This is a major concern due to many factors, including intensifying climate change (e.g., changing rain and evaporation patterns), anthropogenic desertification (due to grazing and the overuse of existing agricultural habitats) and the increasing human population with a rising standard of living. One way towards solve this snowballing problem is to discover genes and pathways associated with abiotic-stress tolerance. Wild relatives of crop plants are a rich source for novel stress-resistance genes. Since wild plants have been dealing evolutionarily with biotic and abiotic stresses for millennia, their genomes contain many genes related to resistance, which can be introduced into domesticated crop species. Traditionally, this has been done through wide crossing and vigorous and continuous breeding schemes. In the past few decades, plant transgenesis has facilitated this process tremendously, saving time, money and resources. In the past 20 years, the Omics age has emerged, allowing the relatively cheap generation of high-throughput data in genomics and metabolomics. One major setback to this revolution is the human inability to manually process this much data. Therefore, many are turning to statistics and computational biology to bridge this data-analysis gap. In this project, we are utilizing the unique genetic materials of the Institute for Cereal Crop Improvement (ICCI) and a Systems Biology approach that brings together physiological experiments (i.e., water use efficiency, osmotic adjustments) with Omics (i.e., genomics, transcriptomics, metabolomics and microbiomics) involving computational and statistical methods (e.g., machine learning and correlation network analysis) to uncover previously unknown genes and pathways associated with resistance to drought and salinity stress in wild and domesticated wheat.  Using the above described approach we have utilized 15 accessions of wild emmer wheat (representative the genetic diversity of wild emmer) and 15 accessions of domesticated wheat (representative the genetic diversity of bread wheat). We plan to further explore this data  to identify shortlist of genes/metabolites/bacteria that might represent a stress specific and/or accession specific response in wheat.  Those genes will be validated using transient (virus induced gene silencing) and stable (tissue culture) transformation in wheat. This research is to the best of our knowledge the first to conduct a system biology approach  in a diversity panel of wild and domesticated wheat.  Additionally, we have utilized a natural variation panel of wild wheat (~450 accessions) to identify single nucleotide polymorphism (SNPs) in grain quality (protein) and quantity  (weight/area). Up to date, we have identified several repetitive and statistically significant SNPs associated with wild wheat seed weight, area and protein content. Moreover, several genes have been identified as putative targets based on function description and expression pattern. For example AMP deaminase (EMBRYONIC FACTOR 1) and B3 domain-containing transcription factor  which are specifically expressed in seed development stages and are putatively involved in seed development,  have been targeted. We are currently in the process of testing those genes using genetic transformation and molecular verification.

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