Date of Graduation

5-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Crop, Soil & Environmental Sciences (PhD)

Degree Level

Graduate

Department

Crop, Soil & Environmental Sciences

Advisor/Mentor

Srivastava, Vibha

Committee Member

Elli, Elvis

Second Committee Member

Drescher, Gerson

Third Committee Member

Egan, Martin

Keywords

Metabolic processes; Phenotypic characterization; Phosphoproteomics; Rice; SnRK1; Transcriptomics

Abstract

SnRK1 is an evolutionarily conserved protein kinase belonging to the SNF1/AMPK family that is composed of a catalytic subunit (α) and two regulatory subunits (β and βγ), and it has a major role in adjusting growth in response to the energy status. SnRK1 phosphorylates and alters the activities of enzymes involved in metabolism and regulates gene expression by altering the activity of chromatin-remodeling enzymes or transcription factors. Rice contains three functional paralogs of SnRK1α: OsSnRK1αA (Os05g0530500), OsSnRK1αB (Os8g0484600), and OsSnRK1αC (Os03g0289100). Although SnRK1 is known to play a central role in plant growth and stress responses, the functional specialization of the SnRK1 α-subunit paralogs in rice remains poorly understood. Using CRISPR/Cas9 mutagenesis to target the functional kinase subunits in rice, we carried out comprehensive phenotypic, transcriptomic, proteomic, and phosphoproteomic analyses of rice snrk1 mutants to investigate the role of SnRK1 signaling in rice stress adaptation, development, and yield. Phenotypic and transcriptomic analyses revealed the role of SnRK1 signaling in controlling growth and stress-related processes in both energy-sufficient and energy-limited conditions and pointed to the subfunctionalization of SnRK1 kinase subunit genes. The snrk1 mutants displayed growth defects under normal and starvation conditions and were more susceptible to Magnaporthe oryzae, where stronger defects were observed in snrk1bc seedlings. Transcriptomic analyses in snrk1 mutants, particularly snrk1bc, reveal a transcriptional profile opposite to that of the wild type. Under normal conditions, the mutant showed upregulation of stress-related and catabolic processes and repression of anabolic pathways. In contrast, under extended darkness, snrk1bc exhibited downregulation of stress-responsive and catabolic processes and inappropriate upregulation of anabolic pathways, indicating that SnRK1B/C plays a major role in regulating the starvation response in rice seedlings. Next, proteomic and phosphoproteomic analyses of snrk1bc seedlings under extended darkness revealed upregulation of growth-related processes, including ribosome biogenesis, cell division, and cellulose biosynthesis, and downregulation of catabolic, energy-generation, and stress-responsive pathways. In addition to the classical protein targets of SnRK1, which showed reduced phosphorylation, phosphoproteomics revealed novel targets, including key components of intracellular membrane trafficking, ethylene signaling, and ion transport. Motif analysis revealed conserved serine-centered phosphorylation motifs characteristic of SnRK1 targets, including the classical SP motif and RxxxS and SxxxL motifs, suggesting that SnRK1 phosphorylation motifs are generally conserved between Arabidopsis and rice. Finally, phenotypic analysis of snrk1 mutants in mature plants showed reduced yield-related traits, such as fewer panicles per plant, lower weight of seeds per plant, and reduced 100-seed weight. Reproductive defects differed among snrk1 mutants, where snrk1a mutants exhibited increased empty spikelets, and snrk1b and snrk1bc showed a higher proportion of shrunken seeds. Shrunken seeds accumulated starch in the endosperm but lacked a visible embryo, suggesting defects in fertilization, embryogenesis, or early seed development. Consistent with these phenotypes, transcriptomic analyses of panicles, pistils, caryopses, and flag leaves revealed misregulation of gametophyte development, pollen, embryo and seed development, carbohydrate metabolism, and reproductive development. Therefore, SnRK1 signaling is essential for rice reproductive development, yield, and grain filling, and SnRK1α paralogs exhibit functional specialization in fertility and grain development.

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