Kethmi Jayawardhane | 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 Kethmi Jayawardhane. This seminar is open to the general public to attend.

Zoom Link: https://ualbertaca.zoom.us/j/92853646605?pwd=LHJenYibqkYSirK33OyVnB0EFB5rx0.1

PhD with Drs. Gavin Chen and Stacy Singer

Thesis Topic: Elucidating novel functions for the AINTEGUMENTA-LIKE 7 (AIL7) transcription factor in plants

Abstract:

Transcription factors (TFs) are crucial proteins that bind to specific DNA sequences in gene promoter or enhancer regions to regulate their transcription, playing pivotal roles in plant growth, development, and survival. Major TF families in plants include APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), Basic Helix-Loop-Helix (bHLH), Myeloblastosis-related (MYB), No-Apical Meristem-ATAF-CUC (NAC), WRKY, and Basic Leucine Zipper (bZIP). Manipulating genes encoding TFs is highly effective for engineering plants, as many of them act as master regulators of many biochemical processes, enabling simultaneous improvements in multiple traits. The AINTEGUMENTA-LIKE 7 (AIL7) gene, a member of the AP2/ERF superfamily, is expressed in overlapping domains with AINTEGUMENTA (ANT) and AIL6 in Arabidopsis thaliana, playing roles in meristem development and maintenance, floral development, and lateral organ positioning. RNA-seq data of AIL7 seed-specific overexpression lines showed upregulation of genes related to abiotic and biotic stress responses and changes in genes related to lipid biosynthesis, compared to wild type plants, suggesting additional physiological roles for this TF.

Thus, the current research is based on the hypothesis that biosynthetic processes related to abiotic/ biotic stress resistance, plant development, and lipid biosynthesis may be improved by constitutively altering the expression of AIL7. Accordingly, this project aimed to evaluate and characterize the precise functions of AIL7 in plant development and stress tolerance in the model plant Arabidopsis, and evaluate the feasibility of using AIL7 as a target to develop crops with better stress tolerance. The research chapters of this thesis focus on the following specific objectives: (1) Generation and characterization of Arabidopsis AIL7 constitutive overexpression plants for their growth performance and tolerance against the biotrophic pathogen Plasmodiophora brassicae, as well as elucidation of the underlying molecular mechanisms associated with sensitivity/ tolerance to P. brassicae; (2) Evaluation of Arabidopsis AIL7 constitutive overexpression lines for temperature stress tolerance and the modulation of membrane lipid composition under stress in these plants, followed by the elucidation of the molecular response of these plants under temperature stress conditions; and (3) Assessment of Arabidopsis AIL7 constitutive overexpression lines and ail7 knockout/ knockdown mutant lines for resilience to drought stress.

To analyze the functions of AIL7 in stress responses, I generated homozygous Arabidopsis lines with constitutive overexpression of AIL7 and characterized ail7 T-DNA mutants through detailed morphological assessments. The AIL7 overexpression lines showed significant increases in plant size, and vegetative biomass, as well as seed size and weight, compared to wild type plants, while the mutants exhibited no notable morphological differences. Subsequently, the resistance of both AIL7 overexpression and mutant lines against P. brassicae was evaluated. Both AIL7 overexpression and knockout, respectively, enhanced tolerance to the pathogen. Targeted gene expression analysis revealed significant alterations in the transcript levels of genes related to pathogen response, particularly in the salicylic acid (SA) and jasmonic acid (JA) defense pathways. Notably, both AIL7 overexpression and mutant lines showed upregulation of SA- and JA-related genes in the absence of P. brassicae, which was confirmed by phytohormone analyses. These findings suggest that phytohormone-mediated defense pathways are constitutively activated in Arabidopsis lines with altered AIL7 expression, whether through overexpression or knockout.

In the second study, AIL7 overexpression and T-DNA mutant lines were evaluated for their resilience under heat- and cold-stress conditions at the seedling stage. AIL7 overexpression plants exhibited better survival capability compared to wild type, while the mutant did not show significant differences only under heat stress conditions but not under cold stress. Moreover, AIL7 overexpression plants demonstrated better growth performance and seed yield under prolonged warming stress during the flowering stage. The inflorescences of AIL7 overexpression plants displayed less hydrogen peroxide accumulation and reduced cell death compared to wild type. As was the case for the clubroot tolerance study, T-DNA mutants also demonstrated enhanced tolerance to heat stress with lower hydrogen peroxide accumulation and cell death in inflorescences. A targeted metabolomics analysis revealed that both AIL7 overexpression and T-DNA mutant inflorescences exhibited a significant increase in total phosphatidylcholine (PC) accumulation under prolonged warming. Interestingly, both monoene and diene percentages of PC were significantly higher in AIL7 overexpression lines whereas mutants did not show a significant difference compared to wild type plants. In addition, AIL7 overexpression inflorescences accumulated significantly more amino acids, which play key roles in stress tolerance, compared to wild type plants. These metabolic changes may contribute to the observed heat stress tolerance. Moreover, gene expression analysis revealed upregulation of several heat shock protein and lipid-related genes in AIL7-overexpression and mutant plants, consistent with their heat stress tolerance capacity.

In the last chapter of the thesis, the drought tolerance capacity of AIL7-manipulated plants was tested. AIL7 overexpression plants performed better under drought stress as they had higher quantum yield, higher chlorophyll content, and better recovery after drought stress. Moreover, AIL7 overexpression plants exhibited higher seed yields compared to wild type plants, suggesting their resilience for water-deficient conditions. The mutant did not produce significant changes compared to wild type control plants under drought stress during seedling or vegetative stages.

Taken together, the characterization of AIL7 revealed that the manipulation of this TF leads to the improvement of several important traits in Arabidopsis simultaneously. In particular, AIL7 overexpression resulted in improved growth traits (plant height, biomass, seed size, and seed yield) and demonstrated better abiotic and biotic stress tolerance. These results indicate that AIL7 would be an attractive candidate for the future molecular breeding of crops.