Developmental and Transcriptional Regulation Underlying the Exceptionally Thick Seed Coat in Narrow- Leafed Lupin (Lupinus angustifolius)

Haihua Lao

doi:10.60867/00000150

Narrow-leafed lupin (Lupinus angustifolius L.) is an emerging high-protein grain legume with increasing relevance for plant-based food systems due to its favourable nutritional profile, low starch content, and environmental sustainability. However, its unusually thick seed coat, constituting approximately 24% of total seed weight, substantially reduces protein yield on a whole-seed basis and limits processing efficiency and consumer acceptance. Understanding the developmental and regulatory basis of seed coat thickening is therefore critical for breeding strategies aimed at improving narrow-leafed lupin as a high-value food protein crop. This thesis investigated the structural, developmental, and transcriptional mechanisms underlying seed coat formation in narrow-leafed lupin through an integrated, multi-scale approach combining developmental anatomy, compositional analysis, and high-resolution transcriptomics across seed initiation and development. Light microscopy and histological analyses established a detailed anatomical framework for seed coat development from pre-anthesis through seed filling and maturation. These analyses demonstrated that the exceptional thickness of the lupin seed coat is primarily attributable to pronounced elongation of the palisade layer and prolonged persistence of the parenchyma region. Comparative histology with soybean revealed that narrow-leafed lupin seed coat initiated integument patterning earlier, maintained maternal tissues for longer, and undergone extended periods of secondary wall deposition. Transient starch accumulation was observed predominantly in parenchyma layers during early developmental stages, while storage protein deposition was largely confined to the cotyledons, providing a compositional context for subsequent molecular analyses. Time-resolved transcriptomic profiling of developing seeds spanning early to late-development stages (3-21 days after pollination, DAP) revealed that narrow-leafed lupin seed development follows a conserved triphasic transcriptional organisation comprising early activation, mid-development stability, and late maturation. However, key phases were temporally reconfigured relative to thinner-coated legumes. Early transcriptional activation was unusually strong and tightly synchronised, while the mid-development phase was markedly prolonged and characterised by sustained expression of pathways associated with cell-wall biosynthesis, phenylpropanoid metabolism, lignin polymerisation, cuticle formation, transport, and signalling. Distinct transcriptional clusters corresponded to major structural transitions, including integument expansion, palisade elongation, parenchyma compression, and the onset of lignification. Multiple transcription factor families, including MYB, ARF, AP2, HD-ZIP, and C2H2 zinc finger proteins, displayed stage-specific expression patterns, indicating coordinated regulation of cell proliferation, radial elongation, and wall fortification. Comparative analysis with soybean highlighted prolonged and delayed integument differentiation in lupin, reflecting fundamental differences in maternal tissue regulation. The earliest developmental window, encompassing pre-pollination to 3DAP, was examined in detail to resolve the initiation of seed coat identity. High-resolution microscopy revealed integument pre-patterning prior to anthesis, confirming strong early maternal control. Transcriptomic analysis of these initiation stages identified auxin-centred hormonal signalling, cytokinin-auxin balance, and abscisic acid-associated pathways as central regulators of early integument growth and differentiation. Key auxin biosynthesis, transport, and signalling genes, including TAA1, PIN7, AIR1, and ARF5, were detected during early seed initiation, together with genes associated with cell-cycle progression, cell-wall remodelling, and starch metabolism. These results indicated that the foundations of seed coat thickness were established within the first 48-72 hours after pollination. Collectively, this thesis provided the first integrated anatomical and transcriptomic framework for seed coat development in narrow-leafed lupin. The findings demonstrated that seed coat thickness raised from early maternal specification, pronounced palisade elongation, persistent parenchyma layers, and sustained secondary wall deposition throughout development. By identifying developmental phase-specific gene modules, phytohormone signalling networks, and candidate transcription factors, this research established developmental timing and prolonged maternal regulatory dominance as the primary determinants of narrow-leafed lupin seed coat architecture. These insights advanced understanding of legume seed biology and provided a mechanistic foundation for future functional studies and breeding strategies aimed at reducing seed coat thickness and enhancing the nutritional and commercial value of narrow-leafed lupin as a sustainable protein crop.

Developmental and Transcriptional Regulation Underlying the Exceptionally Thick Seed Coat in Narrow- Leafed Lupin (Lupinus angustifolius)

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