The role of genes encoding pericycle-specific expression in development of lateral roots and salinity tolerance

Salinity stress is a contributing factor to lowering crop yield due to the accumulation of sodium ions in the shoot and interfering with photosynthesis as well as a range of other independent processes. During the early stages of plant development, lateral roots develop from the pericycle, the cell layer enclosing the vascular tissue. The pericycle is important for processes regulating water and ion transport. Previously, we observed that Arabidopsis high-affinity potassium transporter (HKT1) contributes to salt tolerance and lateral root development. Here, we hypothesize that other genes with pericycle-specific expression are key to lateral root development under salt conditions. Apart from Arabidopsis’ one HKT1 gene, two HKT1 genes (HKT1;1 and HKT1;2) have been identified in tomato, however, their tissue-specific expression has yet to be determined. In this study, we aimed to determine the tissue-specific expression of HKT1;1 and HKT1;2 through cloning their respective promoters into GreenGate expression vectors. In the future, these constructs will be transformed into Agrobacterium rhizogenes and ultimately induce hairy root transformations in tomatoes to localize the pericycle-specific expression of HKT1;2. Additionally, we screened 47 Arabidopsis mutants targeting genes with pericycle-specific expression. The mutants were evaluated for root architecture with and without salt stress. 13 mutants with altered salt stress resilience underwent further screening in PhenoSite Facility at BTI. The results collected within this study will increase our understanding of genes that are specifically expressed in the pericycle, and their contribution to salt stress resilience. This and future studies will further contribute to a more targeted approach to cultivating crops with low-quality water and in areas with no or limited access to freshwater.

I am extremely grateful for the opportunity to work in Magda Julkowska’s lab at BTI and Cornell University this summer. I came to Ithaca lacking a lot of microbiology laboratory experience and I was excited to be out of my comfort zone and to learn everything I could. Over the past ten weeks, my discomfort in new challenges such as cloning via the GreenGate method and the use of R for data analysis has pushed me to become a better biologist. Magda went to great lengths to ensure my experience was the best it could be and it shines in the bittersweet sentiment I am feeling as my time here comes to an end. She has been an amazing mentor; she encouraged me to learn while supporting me and making it okay to make mistakes. My time here has nurtured and simultaneously tested my passion for plant sciences, and I am emerging from it more confident than ever that plant biology is the field I aim to persue.

Molecular Mechanics Underlying Tepary Bean Responses to Drought Stress

As climate change progresses, rising global temperatures continue to increase stress on the world’s agricultural systems. The common bean (whose varieties include green, navy, black, kidney, and pinto beans) has proven particularly susceptible to these changes. In fact, over two-thirds of the world’s legumes are expected to be at risk (Zaulda et al., 2022). The tepary bean, a sister species of the common bean, is indigenous to the southwestern United States and Mexico and has emerged as a viable source of genetic variation for increasing the common bean’s resistance to abiotic stress. Previously, we have identified genes that underlie tepary bean’s resistance to drought stress using forward genetics. Here, we conducted experiments to validate individual genes’ contributions towards drought resilience. Firstly, we successfully validated a protocol for cowpea mosaic virus-induced gene silencing (VIGS) in tepary bean and cowpea. Secondly, we evaluated tepary bean accessions previously classified as stress-resilient or susceptible in the PhenoSight facility for their responses to drought stress. Finally, we screened T-DNA insertion mutants in Arabidopsis of genes homologous to tepary bean genes associated with drought stress resilience. Our results were promising on several fronts. Thanks to our successful validation of the VIGS protocol and evaluation of tepary accessions as tolerant and susceptible to drought stress, we are now targeting drought stress genes using VIGS in tepary beans. Further, our evaluation of Arabidopsis mutants revealed the potential role of several genes in response to osmotic stress at a large evolutionary scale, which may allow us to gain further insight into the broad application of our findings. These and future studies will help us advance our understanding of what makes tepary bean so resilient to drought stress, allowing the identification of future breeding targets in legumes and beyond, and contributing to greater global food security.

I am immensely grateful to have been placed in the Julkowska Lab. The culture of Magda’s lab and the effort she put into her mentorship made me feel welcomed, respected, and safe to make mistakes or not know it all right away. When I arrived, I wrote down three things I hoped to accomplish during this experience: a better understanding of my career goals, to gain confidence, and to increase my knowledge in depth, breadth, and application. I know I have accomplished all three of these goals. This REU has helped me learn about careers in plant science and agriculture I was previously unaware of. The daunting goal of exploring grad school seems somewhat lessened by the information and experience I gained, and I feel much more at home in the lab setting. While no experience is without its challenges, Magda and the BTI staff’s tireless efforts have made the experience worthwhile, and I am truly thankful.

“Exploring salt stress on Arabidopsis thaliana growth with DUF247 genes”

 Project Summary:

Salt stress is detrimental to plant growth. Climate change, floods, irrigation, runoff, and silting all introduce increased amounts of salt into the soil that pose a serious threat to the survival of plants. With our growing population, along with unstable food security, the need for improvements in agriculture is increasing. In a previous study, it was observed that the natural variation in salt stress-induced changes in root-to-shoot ratio corresponds to a locus-encoding domain of unknown function gene, AT3G50160, within the DUF247 family. However, the identified DUF247 locus contains other DUF247 genes that share high sequence similarity to our prime candidate, AT3G50160. Our objective is to determine the physiological relevance of the AT3G50160 gene, as well as its neighboring genes, in response to salt stress by observing the root system architecture on plates and rosette size in soil. By understanding which genes contribute to salt stress tolerance, we can engineer plants that are more resilient to high concentrations of salt.

My Experience:

Throughout my summer at BTI, I was able to better understand what it’s like to work in a research environment and what I would like to do in the future. In such a supportive and friendly lab, with Hayley Sussman as my mentor, I was able to grow with their expertise while also feeling comfortable with making mistakes along the way. I got to improve on relevant skills, such as communication and time management, as well as learn how to use a lot of new research tools, such as ImageJ and R. This internship has allowed me to greatly expand my knowledge and I am looking forward to applying what I learned later on. Having this opportunity to further explore the world of science is an experience I will always be grateful for.

“Hormonal modulation of root system architecture in wild tomatoes under salt stress”

Project Summary:

The increasing human population and reduction in land available for cultivation are threats to agricultural sustainability. This causes environmental stress like soil salinity which alters plant architecture and growth rate, affecting overall plant productivity. Thus, it is imperative to develop more salt-tolerant plants for sustainable agriculture. Since roots are at the interface of saline soil, many root-related architectural traits have been the target of breeding programs for improving crop tolerance and yield. Solanum pimpinellifolium (wild tomato) is the closest relative to Solanum lycopersicum (cultivated tomato) and is an important source for genetic improvement since it goes through adaptation to tolerate any abiotic stress. Previously, Julkowska et al. visually classified four distinct root topologies in terms of the salt stress response by screening root system architecture of 230 S. pimpinellifolium accessions. However, it still remains to be determined 1) how hormonal signaling is involved in creating distinct root topologies, and 2) what is the biological significance of these root topologies in the overall salt stress response. In our experiment, we compared the effect of two classical plant hormones, abscisic acid and ethylene, on the root system architecture of two wild tomato accessions and one cultivated tomato accession. For studying root system architecture, the seedlings are germinated in a control plate for five days and then transferred into treatment plates. Then, the plates start to be scanned for five consecutive days. The images obtained are analyzed through the plugin SmartRoot in the program ImageJ for root tracing, followed by root architectural traits analysis in R.

My Experience:

This experience made me grow both professionally and personally. Coming from such a small island like Puerto Rico and being so far away from home for a whole summer was a real challenge but it was definitely worth it. I made unforgettable friends and memories that forever will live with me. Being an intern at Boyce Thompson Institute certainly changed my vision of what plant biology research is like. It allowed me to enhance my confidence in a research setting, sharpen my critical thinking, learn about new research tools, and develop new skills that will be of high value for my future. In addition to this, I had the opportunity to meet and work with amazing people in my research lab that helped me identify my research interests and define my career goals. This internship provided me with the support and mentorship necessary to approach graduate school applications on the right foot. Overall, I learned valuable lessons and had a fruitful experience that will carry with me.

“Investigation of the role of DUF247 gene in the regulation of salt stress in Arabidopsis Thaliana“

Project Summary:

Salt stress alters plant architecture by impacting the growth and development of different organs, such as main and lateral root growth. Other alterations in plant architecture, such as changes in root-to-shoot ratio are hypothesized to contribute to salt tolerance. Previous screening into salt stress-induced changes in root-to-shoot ratio identified underlying genetic components using Genome Wide Association Study. One of the identified loci was DUF247, encoding a protein with Domain of Unknown Function. The role of DUF247 in regulating root to shoot ratio, as well as general salt tolerance in Arabidopsis is unknown. We aimed to characterize the DUF247 loss-of-function mutant to identify the potential link between this gene and its contribution to the regulation of salt stress response. We compared the duf247 mutant and wild-type (Col-0) plants with or without salt treatment for seed germination and seedling survival rates. Further, we monitored dynamic changes in growth in the above-ground tissues for the soil grown plants for both duf247 mutant and Col-0 plants, and measured the evapotranspiration rate, growth rate as well as rosette fresh weight. Overall, our results showed no significant difference between Col-0 and duf247 mutant plants in terms of germination and survival assays. The future plan will be to repeat the plate experiments using higher concentrations of salt.

My Experience:

My summer internship in the Julkowska lab at BTI has been a fulfilling and extremely valuable experience. My mentor, Maryam Rahmati Ishka, always positively supported me and allowed me to build independence in the lab with confidence in my work. Being welcomed into lab meetings, weekly seminars, and journal clubs provided me with a holistic experience as a plant scientist beyond working at the bench. I learned techniques such as RNA extraction, Real-time qPCR, cDNA synthesis along with other protocols and methods for my experiments. I also enjoyed organizing my results and data onto my poster and preparing to present it. Through the many opportunities to learn about future studies and careers in science, I feel more confident in my future plans for college and am excited to conduct research in the future.