Performing Department
Plant Science & Landscape Architecture
Non Technical Summary
Climate change is intensifying global warming and leading to more frequent and severe droughts, posing a significant threat to the health and survival of our forests. Drought weakens trees, making them more susceptible to diseases, insect infestations, and wildfires (Dale et al., 2001; Kolb et al., 2016). Understanding the molecular mechanisms underlying plant responses to drought is crucial for developing various strategies to improve crop resilience. In this submission, we intend to take advantage of preliminary data generated in the plant model species Arabidopsis thaliana and investigate the role of AT-Hook Motif Nuclear Localized (AHL) genes in drought responses in poplar.The AT-hook motif nuclear-localized (AHL) gene family comprises plant-specific transcription factors that play essential roles in plant growth, development, and stress responses (Aravind and Landsman, 1998; Fujimoto et al., 2004). Recent studies have implicated AHL genes in drought tolerance in various plant species (Zhou et al., 2016; Ambadas et al., 2023; Wong et al., 2024). Preliminary data in Arabidopsis thaliana provide compelling evidence for the involvement of AtAHL20 in drought stress tolerance. Overexpression of AtAHL20 resulted in enhanced drought tolerance in transgenic Arabidopsis plants. RNA sequencing and Gene Ontology (GO) analysis revealed significant upregulation of drought stress-responsive genes in the 35S:AtAHL20 plants, further supporting the positive regulatory role of AtAHL20 in drought tolerance.Building on these promising findings, we aim to functionally characterize the AtAHL20 ortholog in Poplar (Populus trichocarpa), a model tree species with ecological and economic importance. Phylogenetic analysis of the AHL gene family members from Populus trichocarpa and Arabidopsis identified PtrAHL33 (Potri.013G044500) as the closest homolog of AtAHL20 (Wang et al., 2021). Notably, promoter cis-element analysis revealed the presence of three Abscisic acid (ABA)-responsive element (ABRE) binding elements in the PtrAHL33 promoter. ABRE cis-elements are known to bind transcription factors involved in drought stress responses (Yamaguchi-Shinozaki and Shinozaki, 2005), suggesting a potential role for PtrAHL33 in drought tolerance in poplar.There are 37 AHL genes in poplar, categorized into three subfamilies based on conserved domain structures (Wang et al., 2021). Phylogenetic analysis revealed that AtAHL20 and its closest homolog, AtAHL19, cluster with PtrAHL33 (Potri.013G044500) in a distinct clade. Promoter cis-element analysis of PtrAHL33 indicated the presence of three Abscisic Acid (ABA)-Responsive Element (ABRE) binding sites, known to bind transcription factors involved in drought tolerance. In addition, Wang et al. (2021) identified 15 AHL genes in poplar containing the MBS cis-element, which is crucial for managing drought stress. Expression analysis under drought stress and ABA treatment showed significant induction of these genes.This proposal aims to leverage these preliminary findings to investigate the role of PtrAHL33 and other drought-induced AHL genes in poplar. By elucidating the molecular mechanisms and transcriptional networks regulated by PtrAHL genes under drought conditions, this research will provide valuable insights into plant stress responses and inform strategies to enhance crop resilience in the face of climate change.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
0%
Developmental
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Goals / Objectives
The goal of this research is to generate and characterize transgenic lines to test the hypothesis that overexpressing Arabidopsis AtAHL20's orthologue in poplar, PtrAHL33 (Potri.013G044500), and other drought stress-induced PtrAHL genes confers drought tolerance.Objective 1: Functional Characterization of PtrAHL33 and Drought-Stress-InducedPtrAHLGenes in Poplar.Preliminary data from Arabidopsis showed that transgenic lines overexpressing AtAHL20 are drought tolerant (Figure 1). In addition, Wang et al. (2021) recently demonstrated that under both drought and ABA treatments, PtrAHL9, 20, 22, 27, 29, and 34 genes exhibited upregulation in both leaves and roots. To determine whether AtAHL20's orthologue in poplar plays a similar role and whether PtrAHL9, 20, 22, 27, 29, and 34 genes confer drought tolerance, we will use a gain-of-function strategy to characterize these genes in poplar. Due to the high rate of duplication events in the 29-member gene family, functional redundancy exists among multiple AHL genes in Arabidopsis (Zhao et al., 2013), making their characterization by loss-of-function mutagenesis difficult (Zhao et al., 2013; Zhao et al., 2014). Several AHL single loss-of-function mutants, such as ahl6, ahl22, ahl27(esc), and ahl29 (sob3), do not show any phenotypes (Street et al., 2008; Xiao et al., 2009; Zhao et al., 2013; Tayengwa et al., 2020). Therefore, to circumvent problems associated with genetic redundancy, gene overexpression offers an alternative strategy to knockout/knockdown analysis.Objective 2: Phenotypic Analysis of PtrAHL Gain of Function Transgenic Plants Under Drought Stress.We will utilize greenhouse-grown plants and poplar cell cultures to characterize the gain of function and loss of function lines. Greenhouse-grown plants provide a more natural environment, closely mimicking field conditions and allowing for a holistic understanding of drought's impact on whole plants. Greenhouse studies facilitate the study of whole-plant physiology, including root-soil interactions, leaf gas exchange, and overall plant growth and development under drought stress. In addition, greenhouse studies allow for observing phenotypic changes like wilting, leaf curling, and growth reduction, which are vital for understanding drought responses. On the other hand, greenhouse conditions can be inconsistent and influenced by factors like temperature, humidity, and light, leading to variability in results. Suspension cell studies provide a highly controlled and uniform environment, reducing variability and allowing for precise, reproducible experiments. Such uniform conditions enable high-throughput screening of genes, proteins, and metabolites, accelerating the identification of critical regulators and signaling pathways.Objective 3: Comprehensive Transcriptomic Analysis of Drought Stress Response in Poplar.The main goal of this objective is to investigate the transcriptional changes and regulatory mechanisms in poplar under drought stress, leveraging RNA-Seq analysis. We will utilize greenhouse-grown plants to conduct bioinformatics analysis to map out the transcriptional changes and pathways affected by the overexpression of the selected AHL genes. Preliminary data in transgenic plants overexpressing AtAHL20 showed the upregulation of drought response genes AT5G66400/ATD18, AT5G43170/ZFP3, and AT1G77180/SKIP. To examine how overexpression of PtAHL genes affects downstream regulatory pathways and networks, we will use RNA-seq and comparative transcriptomics.
Project Methods
Objective 1: Overexpression analysis ofPtrAHL33and drought-stress-induced PtrAHL genes in Poplar drought response.Methods: Transgenic poplar lines have been and will be constructed using the hybrid clone INRA 717-1B4 (Populus tremula x Populus alba) in the Coleman lab at College Park. PtrAHL33coding sequence was commercially synthesized and subsequently cloned into a pDONR221 entry vector and has since been recombined into the binary vector pH2WG7 via Gateway® reactions. The resulting pH2WG7-PtrAHL33 construct was transformed into Agrobacterium strain GV3101 and then transformed into the hybrid clone INRA 717-1B4. Following co-cultivation, the petiole explants were grown on callus induction media for two weeks before they were transferred to shoot induction media.Similarly, PtrAHL9, 20, 22, 27, 29, and 34 coding sequences will be synthesized using Invitrogen's GeneArtÓ gene synthesis service and cloned into a Gateway-compatible entry vector, pDONR221. After verification by DNA sequencing, the coding sequences will be transferred to the Gateway-compatible binary overexpression vector pH2WG7 via the LR reaction. The T-DNA vector will be transferred to Agrobacterium strain GV3101 and then used to generate transgenic poplars using the Populus tremula x alba 717-1B4 hybrid. After confirmation of increased transcript levels, we will select at least five independent transformed lines for greenhouse studies. Our lab has a robust transformation protocol that routinely yields over 80% transformation efficiency in the INRA 717-1B4 hybrid.Briefly, the method relies upon an initial culture of petiole explants for 7-14 days on callus inducting media to stimulate cell division. Following callus induction, the explants are co-cultivated with Agrobacterium strains containing the genes of interest. Agrobacterium is eliminated after the co-cultivation step, and the explants are further grown on callus-inducing media containing agents (for example, hygromycin) to select transformed cells. Transformed cells are regenerated by transfer to shoot-inducing media, and root induction media induce roots of regenerated shoots. Regenerated plants are then efficiently multiplied by micropropagation. Co-PI Coleman has used this method to produce thousands of transgenic poplars for a suite of genes. Micropropagated shoots quickly acclimate from tissue culture to the greenhouse over 2 weeks and will be transplanted into pots for greenhouse growth studies.Objective 2: Phenotypic Analysis Under Drought StressMethods: We will use greenhouse-grown, potted poplar plants, and suspension cultures for drought stress experiments. The Coleman Lab has extensive experience using cell cultures to study how carbon-nitrogen balance affects cellular growth, metabolism, and the cellular transcriptome (Nargund et al., 2014). Greenhouse-grown plants will be maintained for 8 weeks under controlled environmental conditions (150μmolm−2sec−1; 25°C with a 16-hour light/8-hour dark photoperiod) and automated regular watering until the onset of drought treatments. Drought stress will be imposed on 2-month-old poplar plants by withholding water for 5 days or until visible signs of stress (e.g., leaf wilting) appear. Control plants will continue to receive regular automated watering. Plant tissue samples will be collected after 5 days of treatment, and then subjected toRNA extraction. Samples will be collected immediately before drought treatments for control plants.We will determine the expression levels ofPtrAHL9, 20, 22, 27, 29, 33, and 34in different tissues (roots, leaves, stem) and developmental stages under drought stress treatments using quantitative RT-qPCR. To assess the physiological and morphological impact ofthe overexpression of the selected PtrAHL genes on drought tolerance, we will analyze growth parameters (biomass accumulation, leaf area, and plant height to assess the overall impact of drought on plant growth), water status (relative water content (RWC), leaf water potential, stomatal conductance), photosynthetic performance (chlorophyll fluorescence parameters, chlorophyll content, net photosynthesis rate (Pn) and transpiration rate (E)) and leaf morphology under drought stress.Poplar suspension cells will be established from poplar stem sections from tissue-culture-grown plants, as Nargund et al. (2014) described. Petiole explants will be cultured on callus-inducing media for 7-14 days to stimulate cell division. Following callus induction, the explants will be transferred to 250 ml flasks containing liquid Murashige and Skoog (MS) medium. The suspensions will be subcultured every 7 days in liquid MS medium with 1mgl−12,4-D, 0.1mgl−1NAA, and 0.01mgl−1BA. Typically, 0.4g fresh cells will be transferred to a 250ml flask containing 30ml of liquid MS medium. 5-day-old cell suspensions will be supplemented with 500mM mannitol for 2 or 10h to apply stress. Replicate flasks (n=3) will be harvested, pooled, immediately frozen in liquid nitrogen, and stored at−80°C.Assessment of cellular response to water deficit will be done on growth and viability (cell count and viability, biomass accumulation, growth rate), water status and osmotic adjustment (relative water content, osmotic potential), oxidative stress and antioxidant response (reactive oxygen species and lipid peroxidation measurements).Objective 3: Comprehensive Transcriptomic Analysis of Drought Stress Response in Poplar.Methods: Leaf, stem, and root tissues will be collected from control and drought-stressed greenhouse-grown plants. Total RNA will be extracted from the collected tissues using an RNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions. RNA quality and quantity will be assessed using a spectrophotometer and an Agilent Bioanalyzer.Following the manufacturer's protocol, RNA sequencing libraries will be prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina). After verification of RNA quality, the RNA samples will be submitted to a commercial vendor for RNA-seq. Libraries will be sequenced on an Illumina sequencing platform to generate paired-end reads. The results of RNA-seq will then be used to identify differentially expressed genes and to construct downstream regulatory pathways and networks.