Expression of spider silk protein in tobacco improves drought tolerance with minimal effects on its mechanotype

Spider silk, especially dragline silk from golden silk spiders (Trichonephila clavipes), is an excellent natural material with remarkable mechanical properties. Many studies have focused on the use of plants as biofactories for the production of recombinant spider silk. However, the effects of this material on the mechanical properties or physiology of transgenic plants remain poorly understood. Since glycine-rich proteins play key roles in plants, we evaluated the effects of a glycine-rich spider silk protein on plant mechanical properties (mechanotype) and physiology. We generated tobacco (Nicotiana tabacum) plants producing a nucleus- or plastid-encoded partial component of dragline silk, MaSp1 (major ampullate spidroin-1; MaSp1-tobacco), containing six repetitive glycine-rich and polyalanine tandem domains. MaSp1 accumulation had minimal effect on leaf mechanical properties, but improved drought tolerance. Transcriptome analysis of drought-stressed MaSp1-tobacco revealed the upregulation of genes involved in stress response, antioxidant activity, cellular metabolism and homeostasis, and phenylpropanoid biosynthesis. The effects of drought treatment differed between the nucleus- and the plastid-encoded MaSp1-tobacco, with the latter showing a stronger transcriptomic response and a higher total antioxidant status (TAS). Well-watered MaSp1-tobacco displayed elevated levels of the stress phytohormone ABA, leading to stomatal closure, reduced water loss, activation of stress response, and increased TAS. We show that the moderately enhanced ABA content in these plants plays a pivotal role in drought tolerance, alongside, ABA priming, which causes overall adjustments in multiple drought tolerance mechanisms. Thus, our findings highlight the potential of utilizing glycine-rich spider silk proteins to enhance plant resilience to drought.