Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Forest Resources and Conservation
Non Technical Summary
Current methods of plant improvement are not keeping pace with the needs required by a rapidly growing population, projected to reach 9.8 billion by the year 2050 (FAO), and climate change. To provide enough food and bioenergy, a substantial increase in crop production is required to meet the food and bioenergy demands of future generations, while preserving the world's energy-related and ecological resources. However, agricultural and forestry production continue to be constrained by a variety of abiotic stresses. Drought is one of the most important and widespread environmental stresses that affect plant productivity (Fahad et al. 2017) and is considered a major constraint that increases the risks associated with rain-fed agriculture. Although drought imposes one of the major restrictions on crop yield globally, it remains major challenge to crop production. Therefore, understanding the effect of drought stress on crop's response is critical for developing improved varieties with high productivity to provide enough food and bioenergy to a growing population in a limiting water resources scenario. Fast growing tree species, such as poplars, have high rates of photosynthesis and hydraulic conductance. Consequently, abundant water availability, often provided through intensive irrigation, is critical for their survival and productivity. In these species, drought can lead to hydraulic failure by causing progressive cavitation and conductivity loss of the hydraulic system (Klein et al., 2018). However, distinct poplar genotypes can respond differently to limited water availability. While drought susceptible genotypes experience hydraulic failure, drought tolerant ones display phenotypic plasticity of hydraulic traits that permit rapid acclimation to a changing environment. Physiological mechanisms of drought tolerance are often in conflict with high crop productivity. The hydraulic safety-efficiency framework involves a trade-off between the ability of plants to maximize water transport to achieve high productivity (known as high efficiency) and avoid high levels of drought-induced hydraulic failure (known as safety). This hydraulic trade-off has been observed in several species, wherein plants with larger vessels are more productive at the cost of increased cavitation vulnerability and thus higher drought sensitivity (Nogueira et al. 2020; Rodriguez?Zaccaro and Groover 2019; Hajek et al. 2014) . Nevertheless, several species are known to favor safety mechanisms and modify xylem properties in response to water stress, to mitigate the likelihood of hydraulic failure (Rossi et al. 2013). Although the balance between optimizing hydraulic efficiency with the risk of hydraulic failure can be dramatically altered in response to drought, the genomic and molecular basis of such a trade-off is only sporadically addressed and so is its adaptive value. Wood anatomy and plant hydraulics is central in understanding a species' ability to cope with rapid environmental change. Plants cope with loss of hydraulic conductance induced by embolism through (i) adjustment in the xylem anatomy (Ameglio et al., 2002), (ii) repair of embolized xylem conducts (refilling, Salleo et al. 1996), and (iii) ion-mediate enhancement of residual stem hydraulic conductance ("ionic effect", Zwieniecki, 2001). Therefore, the anatomy of wood and its implications to plant hydraulics are key to understand how plants respond to changes in the environment. Due to their long generation cycle and inability to migrate away from stress, phenotypic plasticity of wood anatomy and plant hydraulics is critical for trees, as it provides a buffer against rapid changes in water availability (Anderegg & Meinzer, 2015). Phenotypic plasticity is genetically controlled, heritable and of potential importance to species' evolution (Nicotra et al., 2010). We hypothesize that, if phenotypic plasticity of wood anatomy and plant hydraulics of specific genotypes is high, they may better adapt to climate shifts, allowing for greater resilience. While loci involved in local adaptation and trait variation have been extensively studied in woody plants, the range and prevalence of phenotypic plasticity and the molecular mechanisms responsible for it are largely unknown. Understanding the mechanisms of wood anatomy and plant hydraulics plasticity is critical for predicting changes in species distribution and productivity under climate change. We recently uncovered a key regulator of vascular development and hydraulic conductivity in poplar (Ribeiro et al., 2020). This newly discovered component of vascular development, named EVE (Enlarged vessel elements), regulates the size and dimension of vessels, affecting photosynthesis and biomass yield (Fig. 1). However, EVE does not affect the susceptibility of xylem conduits to embolism and cavitation. EVE is involved in potassium transport in cambium and differentiating xylem cells. Due the importance of potassium in xylem development and its critical role in drought response, our hypothesis is that EVE is a key player in the ability of poplar to change its hydraulic properties to avoid hydraulic failure under drought regimes. Moreover, we hypothesize that expression of EVE is also important in the mechanism of wood anatomy and plant hydraulics plasticity, playing a role in both adjustment of the xylem anatomy and ion-mediate enhancement of residual stem hydraulic conductance. Finally, based on our preliminary data, wherein trees overexpressing EVE showed larger vessels, higher yield and similar susceptibility to embolism compared to WT trees, we speculate that tress with high expression of EVE may be an exception of the efficiency-safety trade-off. In addition to phenotypic plasticity of the wood anatomy and plant hydraulics, other factors have also been shown to have a positive effect on increasing host stress tolerance . For instance, root endophytes have been shown to have important roles in determining host fitness under periods of drought stress. There is an increasing body of evidence suggesting that root endophytes have important roles in determining host fitness under periods of drought stress and improving trees' drought tolerance (Khan et al. 2016; Lakshmanan et al. 2017; Naylor et al. 2017; Jones et al. 2019). Recent studies have demonstrated that different plant species or even different genotypes of the same species host distinct microbial communities (Horton et al. 2014; Aleklett et al. 2015; Bulgareli et al. 2015; Wagner et al. 2016; Cregger et al. 2018). Moreover, it has also been shown that changes in the host in response to drought, induce changes in the microbiome in plant species, such as grass and rice (Naylor et al. 2017; Santos-Medellín et al. 2017. While the effect of drought on the root microbiome and the effect of the host genome in the microbiome community are been largely explored in some plant species, little is known about the highly complex interaction between the microbiome community and poplar trees. Understanding this complex interaction and further using the ideal tree-microbe symbiosis would allow us to maximize plant growth and increase host drought tolerance. This will further play an important role in improving the economic viability and environmental sustainability of poplar trees as a feedstock. The general objective of this project is to develop forestry crops that are more productive and tolerant to drought, by exploring the phenotypic plasticity of wood anatomy and plant hydraulics of Populus deltoides population as well as uncovering the host genome mechanisms that regulate the establishment of specific endophytes that contribute to drought tolerance.
Animal Health Component
20%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
The general goal of this project is to develop forestry crops that are more productive and tolerant to drought, by exploring the phenotypic plasticity of wood anatomy and plant hydraulics of Populus deltoides population as well as uncovering the host genome mechanisms that regulate the establishment of specific endophytes that contribute to drought tolerance. I propose the following specific ob objectives: 1. Uncover the role and molecular mechanism by which a gene recently identified by us (EVE - Enlarge Vessel Elements) determines poplar's response to drought (through adjustment in the xylem anatomy and/or ionic effect), including its function in phenotypic plasticity and acclimation in response to water stress. 2. Dissect the molecular mechanism of wood anatomy plasticity in response to drought by exploring natural variation of P. deltoides. 3. Evaluate the role of root endophytes in drought tolerance and quantify the host genome's role in regulating the plant endophytic microbiome community in poplar trees. 4. Verify host-microbiome interactions that improve poplar tolerance to drought.
Project Methods
Objective 1 The main goal of the first objective of this project is to uncover the role and molecular mechanism by which EVE determines poplar's response to drought through adjustment in the xylem anatomy, ionic effect and/or hydraulic redistribution. Our expectation is that overexpression of EVE will lead to plants more resilient to water stress. We will start also evaluating the performance of EVE-overexpressing, CRISPS-Cas9 EVE-knockout, and WT trees in response to drought under field conditions. We will initially evaluate the function of EVE in drought resistance by challenging the all the different lines to drought stress (well-watered condition will be used as control). During the course of the experiments, various measurements will be utilized to assess the physiological differences between WT, EVE-overexpressing and CRISPS-Cas9 EVE-knockout poplar trees relative to drought responses, such as net photosynthetic rates, stomatal conductance, intercellular CO2 concentration, leaf water potential (Ψleaf), and leaf are index. At the end of the experiment, biomass of all the trees will also be measured as a response to drought tolerance. To assess the function of EVE in modifying wood anatomy in response to drought we will measure the hydraulic conductivity from the stem of all poplar trees as described in (Ribeiro et al. 2020). Vessel anatomical measurement properties (diameter, area, and count) will be performed on cross-sections of the collected stems as previously described (Ribeiro et al. 2020). In addition, the vessel grouping index, defined by (Carlquist 2001), will be calculated based on cross-sections. Finally, the potassium content of differentiating xylem will be analyzed by Energy-dispersive X-ray spectroscopy. To evaluate the role of EVE in avoiding hydraulic failure through ionic effect we will determine measure vulnerability to cavitation in all genotypes as described in (Sperry et al. 1998; (Gonzalez-Benecke et al. 2010). Moreover, we conduct measurement of ion-mediated enhancement of wxylem hydraulic conductivity in all samples as described in (Jansen et al. 2011). Finally, we will determine the sap electrical conductivity as well as sap potassium concentration using a conductivity meter and atomic emission using a flame photometer, respectively. Objective 2 We previously sampled 579 genetically unrelated individuals of P. deltoides in the central, southern, and eastern U.S., and genotyped them by targeted resequencing (Fahrenkrog et al., 2017b). We have also characterized the xylem transcriptome of 268 individuals from this population and found extensive variation in EVE expression among the individuals. We hypothesize that expression of EVE is a crucial determinant of the phenotypic plasticity of wood anatomy and plant hydraulics in response to water stress. In this objective we will explore the natural variation of a population of P. deltoides using several layers of information (genomics, transcriptomics, and phenomics) to dissect the molecular mechanism of wood anatomy plasticity in response to drought. Experiments will be performed in a greenhouse, as described in objective 1 using with three replicates of 100 individuals of P. deltoides previously selected based on levels of EVE expression (genotypes with low, medium, and high expression). Phenotypic measurements will be performed as described in objective 1. We will transcriptionally phenotype all the 100 individuals. Total RNA will be used for RNA-seq libraries preparation w using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina following the manufacturer's instructions. Ninety-six libraries will be pooled in equal molar proportions and sequenced in paired-end mode (2 × 150 cycles) using the Illumina NovaSeq 6000. For RNA-seq analysis, after read-quality filtering, the pre-processed reads will be aligned to v3.0 of the Populus trichocarpa reference genome using HISAT (Pertea et al., 2016). The expressed transcripts will be assembled, merged, and quantified with StringTie (Pertea et al., 2016) and transcript abundance will be calculated using a "by gene" mixed model. We will use a system genetics approach (by coupling information from co-expression networks and expression quantitative trait loci (eQTL) analyzes) to identify regulatory genes involved in drought response and phenotypic plasticity. Objective 3 In this objective, we want to measure the extent by which the host genome contributes to root microbiome diversity and abundance. Two hundred plants (one hundred genotypes) of P. deltoides will be planted at the Plant Science Research and Education Unit in Citra (Marion Co, FL). It is important to mention that the soil at the Plant Science Research and Education Unit is very sandy and drought-prone, which is an ideal condition for our experiment. The irrigation system will be operated every day in all plots from the planting date for 2 years. After this period, plants will be challenged with water stress, where half of the plants will be selected for irrigation and the other half will serve as non-irrigated, naturally dry controls. One year later, the stem-volume index will be measured as a proxy for biomass yield and drought tolerance (as an indirect effect). In addition, roots will be harvested for microbiome analysis. The community structures and relative abundance of bacterial and fungal endophytes will be quantified using a DNA amplicon sequencing approach. Microbial community composition will be assessed using similar approaches to those employed in our previous studies (Talbot et al. 2014; Chen et al. 2018). We will also estimate the heritability of microbiome properties by treating the estimated bacterial and fungal load, and the relative and absolute species abundance as a phenotype and analyzed using standard quantitative genomics methods for estimating heritability. Furthermore, a genome-wide association analysis will be carried out to detect associations between nuclear SNP markers and the microbiome abundance for each species, one at a time. We will assess the association the host genomic data with each individual taxa (its absolute and relative abundance) using linear regression analysis. Finally, we will quantify the microbiome contribution to drought tolerance. Our hypothesis is that there is a high level of correlation between the functional diversity of the microbiome and disease, with specific species contributing significantly to variation in the plant drought tolerance. We will assess the association of individual taxa (its absolute and relative abundance) with the biomass phenotype (as an indirect response to water stress) using a linear regression analysis. The proportion of the variance associates to the root endophyte microbiome will be estimated using a mixed model analysis. Objective 4 The objective of this objective is to independently verify the discoveries made in the objective 3 and assess our ability to translate these discoveries into practical applications. In order to address this question, we need to evaluate two hypotheses. The first hypothesis to be tested is that (a) host genotype-specific endophytes can be transferred between genetically identical P. deltoides genotypes, and that (b) generalist endophytes can be transferred across distinct P. deltoides genotypes. To test this hypothesis, we will evaluate the ability of rootstock endophytes to colonize sterile scions. The second hypothesis to be tested in this aim, is that specific endophytes can modify drought tolerance phenotypes. To test the second hypothesis, we will graft scion from genotype shown to be more sensitive to drought into rootstock from plants that contain genotype-specific and generalist endophytes predicted to be modifiers of drought tolerance. The completion of this objective will potentially establish an approach for improving poplar tolerance to drought by exploiting the interaction between plant and microbiome.