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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Hannoufa, Abdelali

2nd Supervisor

Kohalmi, Susanne

Co-Supervisor

Abstract

The highly conserved plant microRNA156, miR156, affects various aspects of plant development and stress response by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. Our understanding of the role of miR156 and its mode of action in alfalfa’s (Medicago sativa L.) response to drought and flooding is still elusive, and thus this study was aimed at filling this gap in knowledge. Physiological parameters, metabolite and transcriptional analyses showed an interplay between miR156/SPL13 and WD40-1/DFR to mitigate drought stress. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune the DIHYDROFLAVONOL-4-REDUCTASE (DFR) level for enhanced anthocyanin biosynthesis. Moreover, RNAseq-derived weighted gene co-expression network analysis (WGCNA) of SPL13RNAi alfalfa plants showed tissue-and genotype-specific drought responses. Accordingly, transcripts mediating stress-mitigating metabolites, such as anthocyanin, were increased in stem tissues of drought-stressed plants, while those involved in photosynthesis were maintained in leaves. Moreover, drought-stressed roots showed elevated transcripts associated with metal ion transport, carbohydrate, and primary metabolism.

The role of miR156 in flooding tolerance was also investigated using flooding-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars, along with miR156OE and SPL13RNAi plants. Additionally, to examine the role of ABA and SnRK1 in regulating miR156 expression, ABA insensitive (abi1-2, abi5-8) Arabidopsis thaliana mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) SnRK1 were used. Investigation of physiological parameters, hormone profiling, and global transcriptomics showed a positive role for miR156 in flooding tolerance, and a comparison of Arabidopsis mutants and transgenic lines showed that miR156 expression was affected by SnRK1 to enhance anthocyanin and ABA metabolites. Transcriptomics analysis also revealed nine new alfalfa SPLs, three of which responded to flooding (SPL7a, SPL8, and SPL13a) along with the previously identified SPL4, SPL9, and SPL13.

Characterization of the newly identified SPLs, along with understanding the mode of action of miR156 in alfalfa’s response to drought and flooding, will provide useful tools in marker-assisted breeding of alfalfa and resource to scientific knowledge.

Summary for Lay Audience

Among the various abiotic stresses, drought and flooding are two extremes of water availability affecting the production and productivity of agricultural crops, including alfalfa (Medicago sativa L.). The frequency, distribution, and intensity of drought and flooding are increasing in conjunction with the current climate change phenomenon, highlighting the need for developing tolerant cultivars. The highly conserved plant microRNA156, miR156, affects various aspects of plant development and stress response by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. Our understanding of the role of miR156 and its mode of action in drought and flooding tolerance in alfalfa is limited, and thus this study is aimed to fill this gap.

In the current study, the role of miR156 and its mode of action in regulating drought and flooding tolerance was investigated. Hence, alfalfa plants with increased levels of miR156 and altered miR156-regulated downstream genes (such as SPL13) were used to assess parameters linked to abiotic stress. The physiological and molecular responses revealed the positive role of miR156 in drought and flood stress tolerance. Upon drought stress, alfalfa plants with low to moderate level miR156 maintained plant water status and physiological activity by increasing responsible genes and stress-reducing metabolites. On the other hand, plants containing miR156 at higher levels coordinated genes to control membrane permeability, increase stress-reducing metabolites, keep physiological activity, and increase abscisic acid in response to flooding. The flooding experiment also identified nine new SPLs to be exploited in future studies. Knowledge gained on the role of miR156 and its target SPL genes in response to both drought and flooding will be utilized in developing tools for alfalfa breeding and as a resource for scientific knowledge.

Creative Commons License

Creative Commons Attribution-Share Alike 4.0 License
This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License.

Available for download on Saturday, January 01, 2022

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