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

Abstract

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.

Keywords: ABA; drought tolerance; glycine‐rich; mechanotype; spider silk; tensile strength.

Figure 1

Generation of MaSp1‐expressing transgenic and transplastomic tobacco. (a) Approach for expressing a transgene encoding the MaSp1 repeat domain (6×) from the nuclear and plastid genomes of N. tabacum. (b) Schematic diagram of the modified vector pBI121_m used for the nuclear expression of MaSp1 in tobacco, which was confirmed by (c) PCR genotyping and (d) immunoblotting of 7 μg of total soluble protein (TSP) per well from the T0 transgenic tobacco shoots using a monoclonal anti‐6 × His antibody. (e) Schematic diagram of the modified vector pPRV112A_m used for the plastid expression of MaSp1 in tobacco, which was confirmed by (f) PCR genotyping of T0 and (g) T1 progeny, and (h) southern blotting of 10 μg genomic DNA from T1 and T2 progeny of #2–2 (hereafter MaSp1 _pla #2–2) using a DNA probe targeted to the trnV–16SrDNA region following restriction digestion by SacII. Digestion with SacII yielded approximately 1.6 kb fragment in WT and approximately 3.3 kb fragment in the transplastomic lines with P _psbA :MaSp1:T _rps16 and P _rrn :aadA:T _psbA insertion. Protein expression was confirmed by (i) immunoblotting of 22–23 μg of TSP per well from the T2 progeny of transplastomic tobacco shoots using a monoclonal anti‐6 × His antibody. CDS, coding sequence. Positive bands or signals of the expected sizes are indicated by black arrows. Dimerized MaSp1 in (h) is indicated by a blue arrow.

Figure 2

Tensile test of leaves of WT, MaSp1 _nuc #1, GFP _nuc , MaSp1 _pla #2–2, and GFP _pla plants with varying moisture contents. Representative photographs of plants used for the tensile test at (a) 85% and (b) 15% leaf moisture. (c) Tensile strength, (d) strain at break, (e) Young's modulus, and (f) toughness of leaves at 85% and 15% leaf moisture content. Stress–strain curves of representative leaf sections of WT, MaSp1 _nuc #1, GFP _nuc , MaSp1 _pla #2–2, and GFP _pla with (g) 85% and (h) 15% leaf moisture contents. Data represent means ± SEM of n = 12 to 20 leaf sections from 4 individual plants. No significant difference in values between the genotypes was observed using one‐way ANOVA (Tukey's test). Representative data closest to the average values for each line are shown in the stress–strain curves. n.d. indicates no data.

Figure 3

Drought tolerance and recovery of MaSp1‐tobacco. MaSp1‐tobacco were grown in (a–c) PEG‐containing medium at (a), (b) ψ_w − 1.42 MPa and (c) ψ_w − 0.63 MPa and in (d), (e) soil. (a) Single seedling dry weight of WT, MaSp1‐expressing plants (#1_nuc and #2‐2_pla), and their corresponding GFP controls (GFP _nuc and GFP _pla ) before drought treatment (0 days after drought induction [DAD]) and 8 days after recovery (DAR) (n = 12 to 40). (b) % decrease in seedling dry weight due to drought at 2 DAR, obtained by comparing the weights of the respective lines grown under unstressed conditions for the same period of time (n = 12–40). (c) Increase in single seedling dry weight during drought at 9 DAD compared with 0 DAD (n = 12–16). (d) Growth of 19‐day‐old WT and MaSp1‐expressing plants (#1_nuc and #2‐2_pla) on soil under 16 days of water withholding treatment and after recovery. The magnified image shows increased growth and recovery of MaSp1‐expressing plants under water withholding treatment from a representative pot. (e) Increase in seedling dry weight under water withholding treatment at 2 days after rewatering relative to the weights of the 18‐day‐old seedlings before drought initiation (n = 40). Data represent means ± SEM. Asterisks indicate significant differences at * P < 0.05, ** P < 0.01 and *** P < 0.001, as determined by one‐way ANOVA (Tukey's test).

Figure 4

RNA‐seq and total antioxidant status of MaSp1‐tobacco grown at ψ_w − 0.63 MPa. (a) Differentially expressed genes (DEGs) with more than twofold differences in expression between drought‐stressed WT, MaSp1 _nuc , and MaSp1 _pla , where a higher number of DEGs was observed in MaSp1‐expressing plants with MaSp1 _pla  > MaSp1 _nuc . (b) Gene set enrichment analysis (GSEA) of 10 highly enriched GO terms with more than twofold differences relative to the WT. (c) Higher total antioxidant status of MaSp1‐expressing plants (MaSp1 _pla  > MaSp1 _nuc ) compared with WT for n = 12–20 plants, where each sample point corresponds to the average value of four plants from different culture plates. Data represent means ± SEM. Asterisks indicate significant differences at *P < 0.05 and ** P < 0.01, as determined by one‐way ANOVA (Tukey's test). RNA‐seq data were obtained for pooled RNA from n = 8 plants per genotype.

Figure 5

Moderate increase in ABA content of well‐watered MaSp1‐tobacco leads to drought tolerance. ABA content in the shoots of WT and MaSp1‐tobacco under (a) drought stress (14‐day‐old seedlings in soil–water withheld for 22 days without recovery), and (b) in well‐watered conditions (37‐day‐old seedlings in soil) from n = 7 groups of 3 plants each. (c) RT‐qPCR of genes involved in (top panel) ABA biosynthesis (9‐cis‐epoxycarotenoid dioxygenase (NCED3) and Xanthoxin dehydrogenase (ABA2)), (middle panel) ABA signaling (Serine threonine protein phosphatase 2C (PP2C) and Mitogen‐activated protein kinase kinase (MAPKK9)) and (bottom panel) ABA catabolism (cytochrome P450 (CYP707A1)), expressed as relative abundance to Actin in shoots from n = 15 plants, where the gene/Actin ratio for WT was set to 1. (d) Approximately 30‐day‐old plants were observed by infrared camera at 23°C. The top panel shows infrared images, and the bottom shows photos of plants. Bars indicate 2 cm. (e) The surface temperature of the leaves was analyzed using an infrared camera. Data were obtained from six independent plants. (f) Total antioxidant status (TAS) in shoots from non‐stressed MaSp1 _nuc and MaSp1 _pla and the WT (n = 4–7 plants). (g) Leaves from approximately 30‐day‐old plants were detached and placed at 23°C, and their weights were measured at the beginning, 1 and 2 h later. Data were obtained from 12 independent plants and represent means ± SEM. Asterisks indicate significant differences at * P < 0.05, ** P < 0.01, and **** P < 0.0001 using one‐way ANOVA (Tukey's test).

Figure 6

Transcriptional regulation of genes in drought‐stressed (ψ_w − 0.63 MPa) MaSp1‐tobacco with previously reported roles in stress tolerance. (a) Heatmap of DEGs (more than twofold difference in expression relative to the WT) which are responsive to ABA or stress and previously implicated in stress tolerance in tobacco or other plant species as shown in Table S2, and (b) RT‐qPCR confirmation of few key players in drought tolerance (shown in red in (a)), expressed as relative abundance to Actin in shoots from n = 15 plants, where the gene/Actin ratio for WT was set to 1. Data represent means ± SEM.