Udaya Subedi | ALES Graduate Seminar

Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES PhD Final Exam Seminar by Udaya Subedi. This seminar is open to the general public to attend.

Zoom Link: https://ualberta-ca.zoom.us/j/91031969840?pwd=SX4NDt49WmRoALAHMupk21BjR9m82i.1

PhD with Drs. Gavin Chen and Stacy Singer

Thesis Topic: Improving abiotic stress tolerance in alfalfa through the down-regulation and genome editing mediated knockout of multiple genes

Abstract:

Two different water extremes, drought and waterlogging, exert profound detrimental impacts on productivity and yields of alfalfa. With climate change intensifying globally, these abiotic stressors are projected to escalate in prevalence, exacerbating agricultural challenges. To minimize existing losses, there is a critical need for the development of new alfalfa cultivars that can better withstand these types of stresses; however, progress has been hindered by the complexity of stress tolerance mechanisms, as well as significant gaps in our understanding of these processes. As such, I sought to provide further insight into the mechanisms behind resilience to waterlogging and drought stresses in alfalfa through the assessment of genotypes in which two transcription factors, WUSCHEL-RELATED HOMEOBOX 13-2 (MsWOX13-2) and TELOMERASE ACTIVATOR 1/REGULATOR OF SYMBIOSOME DIFFERENTIATION (MsTAC1/RSD), had been down-regulated, respectively. Both of these genes had been implicated in abiotic stress responses previously; however, their roles in alfalfa had yet to be elucidated.

In the case of alfalfa MsWOX13-2, which I found to be expressed preferentially in roots and differentially under waterlogging stress, the RNAi-mediated down-regulation of MsWOX13- 2 in alfalfa had no effects on growth or morphological characteristics under control (wellwatered) conditions. However, under waterlogged conditions, MsWOX13-2 RNAi plants exhibited enhanced performance, as evidenced by a reduced impact of stress on morphology and greater survivability compared to empty vector genotypes. In addition, MsWOX13-2 RNAi genotypes exhibited an apparent reduction in leaf chlorosis under waterlogging, which correlated with higher chlorophyll retention and maximum quantum efficiency of photosystem II (Fv/Fm), compared to empty vector genotypes. This reduction in stress symptoms may be linked, at least in part, with the fact that MsWOX13-2 RNAi leaves accumulated less malondialdehyde (MDA), which is a marker for oxidative stress, and displayed higher superoxide dismutase (SOD) activity. RNA-Seq analysis confirmed the presence of differentially expressed genes (DEGs) related to photosynthesis, antioxidant activities, anaerobic respiration, cell wall modulation, phytohormone-related pathways, and transcription factors. Subsequently, CRISPR/Cas9 was used to edit the MsWOX13-2 gene in alfalfa with up to two alleles mutated in the first generation, resulting in bi-allelic mutations that conferred waterlogging tolerance and enhanced branching.

Alfalfa MsTAC1/RSD, on the other hand, which shares orthology with Arabidopsis TAC1 and Medicago truncatula RSD, was highly expressed in nodules in a manner similar to MtRSD and was up-regulated as drought stress progressed. Under well-watered conditions, MsTAC1/RSD RNAi genotypes had equivalent biomass production with smaller stem diameter and delayed flowering, as well as increased lateral root branching. Under drought conditions, these genotypes exhibited improvements in their resilience to water-deficit compared to empty vector control genotypes as evidenced by higher plant height and dry root biomass following drought recovery. This tolerance to drought could be attributed, at least in part, to a reduction in stomatal density, and a consequent decrease in leaf water loss, along with improved baseline total antioxidant capacity and higher superoxide dismutase (SOD) and catalase (CAT) activity under drought stress. In line with this, transcript profiling via RNA-Seq demonstrated changes in the expression of genes related to photosynthesis, antioxidant activities, cell wall modulation, phytohormones, and transcription factors, which could have contributed to the observed improvements in MsTAC1/RSD RNAi genotypes under drought stress. Additionally, MsTAC1/RSD RNAi genotypes exhibited shorter telomeres, as evidenced by lower relative telomere length, and showed impaired nodulation, with fewer pink nodules compared to wild-type genotypes. Enhanced drought resilience and impaired nodulation were further confirmed through CRISPR/Cas9-mediated knock-out of the MsTAC1/RSD gene in alfalfa.

Taken together, these results indicate that MsWOX13-2 and MsTAC1/RSD function as negative regulators of waterlogging and drought stress response in alfalfa, respectively. However, MsTAC1/RSD seems to be positively associated with telomere maintenance and nodulation, suggesting its multiple roles in plant development and stress response in alfalfa. While MsTAC1/RSD CRISPR genotypes will not provide a beneficial source of germplasm due to the fact that these genotypes do not produce nitrogen-fixing nodules, transgene-free genome edited MsWOX13-2 CRISPR genotypes could be ideal candidates for improving alfalfa productivity under adverse environmental conditions in the future.