Plant material

WT N. tabacum cv. “Petit Havana” seeds carrying TMV resistance (NN) were a gift from Professor Spencer Whitney. Lines exhibiting increased or reduced expression of PsbS were generated within this study as described below.

Recombinant DNA and transformation

The N. benthamiana PsbS gene coding sequence (www.uniprot.org, Q2LAH0_NICBE) was cloned in between the cauliflower mosaic virus 35S and octopine synthase terminator in the pEARLYGATE 100 binary vector. The resulting binary vector pEG100-NbPsbS conferred microbial resistance to kanamycin and bialaphos resistance in planta (Supplementary Fig. 1). Nicotiana tabacum cv. “Petite Havana” was transformed with pEG100-NbPsbS using the Agrobacterium tumefaciens-mediated protocol33. Copy number and homozygosity were assessed using digital droplet PCR34. Results shown are for homozygous offspring unless otherwise described.

Transcription and protein expression

Five leaf discs (total 2.9 cm2) were from the youngest fully expanded leaf of five plants per genotype (controlled conditions) or four plants per genotype (field). Samples were taken 2 h after the start of the photoperiod. Protein and mRNA were extracted from the same leaf sample (NucleoSpin RNA/Protein kit, REF740933, Macherey-Nagel GmbH & Co., Düren, Germany). Extracted mRNA was treated by DNase (Turbo DNA-free kit; AM1907, Thermo Fisher Scientific, Waltham, MA, USA) and transcribed to cDNA using Superscript III First-Strand Synthesis System for RT-PCR (18089-051, Thermo Fisher Scientific, Waltham, MA, USA). Quantitative reverse transcription PCR was used to quantify PsbS transcripts (5′-GGCACAGCTGAATCTTGAAAC-3′ and 5′-CAGGGACAGGGTCATCAATAAA-3′) relative to NtActin (5′-CCTCACAGAAGCTCCTCTTAATC-3′ and 5′-ACAGCCTGAATGGCGATATAC-3′) and NtTubulin (5′-GTACATGGCCTGTTGTTTGATG-3′ and 5′-CTGGATGGTCCTCTTTGTCTTT-3′).

Total protein concentration was quantified using a protein quantification assay (ref. 740967.50, Macherey-Nagel GmbH & Co., Düren, Germany). Samples containing 1 µg total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis electrophoresis, blotted to membrane (Immobilon-P, IPVH00010, Millipore, Tullagreen, Carrigtwohill, Ireland) using semi-dry blotting (Trans-Blot SD, Bio-Rad, Hercules, CA, USA), and sequentially immuno-labeled with primary antibodies raised against AtPsbS (1:2,000 dilution; AS09533, Agrisera, Vännäs, Sweden) and AtPsbO (1:20,000 dilution; AS06142-33, Agrisera, Vännäs, Sweden) followed by incubation with secondary antibodies (1:2,500 dilution; W401B, Promega, Madison, WI, USA). The sequential use of the two primary antibodies was verified empirically against blots where only one antibody was used and dilution series were used to establish the quantifiable range. Chemiluminescence was detected using a scanner (ImageQuant LAS-4010, GE Healthcare Life Sciences, Pittsburgh, PA, USA). A protein ladder (Precision Plus Protein Kaleidoscope Prestained Protein Standards, #1610375, Bio-Rad, Hercules, CA, USA) was used as a size indicator on each gel. Protein bands were quantified using densitometry with ImageQuant TL software (version 7.0 GE Healthcare Life Sciences, Pittsburgh, PA, USA) (Data Set 2 and 11 in Data Repository; https://data.mendeley.com/datasets/nsbjps9rkg/draft?a = 10508d31-685a-4a62-8f96-cb591c569e97). PsbS expression was normalized based on PsbO bands.

Photosynthetic gas exchange under controlled conditions

Seedlings of psbs-4, PSBS-43, PSBS-28, and WT were germinated on growing medium (LC1 Sunshine mix, Sun Gro Horticulture, Agawam, MA, USA) in a controlled-environment walk-in growing chamber (Environmental Growth Chambers, Chagrin Falls, OH, USA) with photoperiod set to 12 h and temperature controlled at 23/18 °C (day/night). Five days after germination, psbs-4 seedlings with low NPQ were identified through chlorophyll fluorescence imaging and together with PSBS-28, PSBS-43, and WT seedlings transplanted to 3.8-L pots and randomly positioned in a controlled-environment chamber (PGC20, Conviron, Winnipeg, MB, Canada) with photoperiod set to 16 h and air temperature controlled at 20/25 °C (night/day). Light intensity at leaf-level was controlled at 500 µmol m−2 s−1. Plants were watered and plant positions were repositioned at random every 2 days until the fifth leaf was fully expanded. Gas exchange measurements were performed using an open gas exchange system (LI6400XT, LI-COR, Lincoln, NE, USA) equipped with a 2-cm2 leaf chamber and integrated modulated fluorometer. All chlorophyll fluorescence measurements were performed using the multiphase flash routine35. To determine the light response of A n and whole-chain photosynthetic electron transport, gas exchange and pulse amplitude-modulated chlorophyll fluorescence were measured at a range of light intensities. Block temperature was controlled at 25 °C, [CO 2 ] inside the cuvette was maintained at 380 µmol mol−1 and leaf-to-air water VPD was controlled to 1.1–1.4 kPa. Leaves were clamped in the leaf cuvette and dark-adapted for 1 h, after which minimal (F o ) and maximal fluorescence (F m ) were measured to determine maximal efficiency of whole-chain electron transport36 (F v /F m , Eq. 1)

$$\begin{array}{*{20}{c}} {{F_{\mathrm{v}}}/{F_{\mathrm{m}}}} & = & {({F_{\mathrm{m}}} - {F_{\mathrm{o}}})/{F_{\mathrm{m}}}} \end{array}.$$ (1)

Subsequently, light intensity (100% red LEDs, λ peak 630 nm) was slowly increased from 0 to 50, 80, 110, 140, 170, 200, 300, 400, 500, 600, 800, 1,000, 1,500, and 2,000 µmol m−2 s−1. When steady state was reached, A n , g s , and C i were logged, and F′ and F m ′ were measured to estimate the operating efficiency of whole-chain electron transport36 (F q ′/F m ′, Eq. 2). Since stomatal movements can include very long-term diurnal components37,38, our routine was aimed at measuring only relatively short-term stomatal responses to changes in light intensity, and steady-state waiting times were kept between 10 and 20 min per step. NPQ of chlorophyll fluorescence was determined according to Eq. 3 assuming a Stern–Volmer quenching model39. Minimal fluorescence without dark adaptation (F o ′) was also determined (using a short far-red pulse to fully oxidize Q A ). The fluorescence parameter qL (Eq. 4) was used to estimate the fraction of Q A in its oxidized state (and correspondingly, Q A redox state as 1 – qL). The derivation of this parameter is assuming a “lake” model for photosynthetic antenna complexes (i.e., antennae are shared between reaction centers)40:

$$\begin{array}{*{20}{c}} {F_{\mathrm{q}}}{^{\prime}/{F_{\mathrm{m}}}^{\prime}} & = & {({F_{\mathrm{m}}}^{\prime} - {F^{\prime}})/{F_{\mathrm{m}}}^{\prime}} \end{array},$$ (2)

$$\begin{array}{*{20}{c}} {{\mathrm{NPQ}}} & = & {{{F_{\mathrm{m}}}/{{F_{\mathrm{m}}}^{\prime}}}} \end{array} - 1,$$ (3)

$$\begin{array}{*{20}{c}} {{\mathrm{q}}_{\mathrm{L}}} & = & {(1/{F}^{\prime} - 1/{F_{\mathrm{m}}}^{\prime})/(1/{F_{\mathrm{o}}}^{\prime} - 1/{F_{\mathrm{m}}}^{\prime})} \end{array}.$$ (4)

To evaluate the CO 2 response of A n , leaves were allowed to reach steady state at a light intensity of 2,000 µmol m−2 s−1 (100% red LEDs, λ peak = 630 nm), with block temperature controlled at 25 °C and [CO 2 ] in the airstream set to 400 µmol mol−1. Subsequently, [CO 2 ] was varied from 400 to 300, 200, 100, 75, 400, 400, 500, 600, 700, 800, 1,000, 1,200, and 1,500 µmol mol−1. When steady state was attained, A n , g s , and C i were logged. V cmax was determined from the response of A n to chloroplastic CO 2 concentration (C c ) by fitting a biochemical model41 with temperature corrections42 to measurements. C c required an estimate of mesophyll conductance to CO 2 transfer (g m ). This was estimated independently for each point in the CO 2 response curve from parallel chlorophyll fluorescence measurements according to the variable J method43. J max was determined by fitting a non-rectangular hyperbola to light response curves of linear electron transport estimated from chlorophyll fluorescence44. Stomatal limitation of A n was computed using measurements at ambient CO 2 (C a = 380 μmol mol−1) and saturating light intensity, and predicted values of A n when stomata are not limiting (i.e., C i would equal C a )44.

Rubisco activation state and content

Plants were grown under controlled conditions as described above. Youngest fully expanded leaves were clamped in the cuvette of an open gas exchange system (LI6400XT with 2 × 3 LED light source), with light intensity set to 1800 μmol m−2 s−1, CO 2 concentration set to 400 μmol mol−1, and block temperature set to 25 °C. After steady-state gas exchange was reached, leaves were rapidly removed and a disc of 0.55 cm2 from the center of the portion of the leaf that had been enclosed in the cuvette was snap frozen in liquid N. Rubisco activity was determined by the incorporation of 14CO 2 into acid-stable products at 25 °C following an existing protocol45. Samples were ground in tenbroek glass homogenizers with ~2 mL cm−2 CO 2 -free extraction buffer containing 100 mM Hepes-KOH (pH 7.5), 2 mM Na 2 ethylenediaminetetraacetic acid (EDTA), 20 mM MgCl 2 , 5 mM dithiothreitol (DTT), 5 mg mL−1 polyvinyl pyrrolidine, 15 mM amino-n-caproic acid and 3.5 mM benzamidine, and 5% v/v protease inhibitor cocktail (P9599, Sigma, St. Louis, MO, USA). Within 30 s of extraction, samples were assayed for initial Rubisco activity in a buffer containing 100 mM Bicine-NaOH (pH 8.2), 1 mM Na 2 EDTA, 20 mM MgCl 2 , 5 mM DTT, 1 mM ATP, 0.5 mM ribulose-1,5-bisphosphate, and 12.8 mM NaH14CO 3 (15 Bq nmol−1, Vitrax, Placentia, CA, USA). Assays were run for 30 s and terminated with the addition of 300 μL 5 N formic acid. The radioactivity of acid-stable products was determined by liquid scintillation counting (Packard Tri-Carb 1900 TR, Canberra Packard Instruments Co., Downers Grove, IL, USA). After determining initial activity, the extract was incubated with 10 mM NaHCO 3 and 20 mM MgCl 2 for 20 min at room temperature, and the total activity of the extract was assayed as above. Unless stated otherwise, all other reagents were purchased from Sigma (St. Louis, MO, USA). Purified RuBP was used in both initial and total activity assays to avoid underestimating the activation state46. The activation state of Rubisco is determined by the ratio of initial to total activity. Rubisco content was determined from carbamylated samples extracted as above using a [14C]carboxy-arabinitol bisphosphate-binding assay47 with a specific activity of 583 Bq nmol−1 Rubisco, assuming eight binding sites per Rubisco45.

Stomatal density and stomatal complex dimension

Plants were grown under controlled conditions as described above. Fresh leaf samples were taken from the youngest fully expanded leaf and mounted onto a microscope slide using double-sided tape. Topographies of the adaxial and abaxial surfaces were measured using a μsurf explorer optical topometer (Nanofocus, Oberhausen, Germany). The 20×/0.60 objective lens (image size 0.8 × 0.8 mm2) and the 50×/0.80 objective lens (image size 0.32 × 0.32 mm2) were used, respectively, for stomatal density quantification and measurements of stomatal complex dimensions. Based on a prior bootstrap analysis, 8 and 10 images were analyzed for each of four biological replicates for stomatal density quantification and measurements of stomatal complex dimensions, respectively. For stomatal density quantification, raw images were first optimized for light contrast in μsoft analysis software (Nanofocus, Oberhausen, Germany) and then exported into TIF format. Each stoma was labeled and counted manually using multi-point function in ImageJ (ImageJ 1.51k, NIH, Rockville, MD, USA). Stomatal density was derived by dividing the number of stomata in each image by the image size (0.64 mm2). To measure the length and width of the stomatal complex, the distance measurement function in μsoft analysis software was applied on each stoma which could be completely observed in the image. Lines were drawn manually from end to end of the stomatal complex ellipse to measure stomatal length and width.

Seedling propagation for field experiment

Homozygous T 2 (psbs-50 and PSBS-28) or T 3 (PSBS-34, PSBS-43, and PSBS-46) seeds as well as segregating T 1 seed from psbs-4 and WT seed from the same harvest date were sown in the greenhouse on May 16, 2016. Five days after germination, seedlings exhibiting severely reduced NPQ were identified by chlorophyll fluorescence imaging of the psbs-4 T 1 progeny. These low NPQ seedlings as well as seedlings from all other lines and WT were propagated hydroponically for 2 weeks in floating trays (Transplant Tray GP009 6 × 12 cells, Speedling Inc., Ruskin, FL, USA) filled with specialized growing medium for hydroponics (Pro-mix PGX, Premier Tech, Quakertown, PA, USA). The concentration of total dissolved solids in the solution was measured every 2 days with a handheld TDS meter (COM-100, HM Digital Inc., Culver City, CA, USA) and adjusted to 100 ppm by the addition of 20-10-20 water-soluble fertilizer (Jack’s Professional, JR Peters Inc., Allentown, PA, USA). Five days after the transplant to trays, Etridiazole fungicide (Terramaster 4EC to a final concentration of 78 μl L−1, Crompton Manufacturing Company Inc., Middlebury, CT, USA) was added to the solution to protect the plants against root fungus disease in the field. Two applications of Mancozeb (Dithane Rainshield Fungicide at 1 g L−1, Dow AgroSciences Canada Inc., Calgary, AB, Canada) were applied 6 and 9 days after transplant to prevent foliar fungus disease. On the same days, seedlings were sprayed with fermentation solids and solubles from Bacillus thuringiensis, subsp israelensis, strain AM65-52 (Gnatrol WDG Biological larvicide at 1 mL L−1, Valent Biosciences Corp., Libertyville, IL, USA) to reduce the greenhouse population of fungus gnats.

Field experimental design

Seedlings were transplanted to an experimental field site at the University of Illinois Energy Farm (40.11°N, 88.21°W) on June 9, 2016. The field was prepared 2 weeks prior to transplant by rototilling, cultivation, and harrowing. At this time, chlorpyrifos (1.5 g m−2 Lorsban 15 G Insecticide, Dow AgroSciences Canada Inc., Calgary, AB, Canada) was worked into the soil to suppress cutworm damage, sulfentrazone (29 μL m−2 Spartan 4 F preemergence herbicide, FMC Agricultural Solutions, Philadelphia, PA, USA) was applied to reduce the emergence of weeds and slow-release fertilizer (30.8 g m−2 ESN Smart Nitrogen, Agrium US Inc., Denver, CO, USA) was put down. After transplant, all seedlings were sprayed with thiamethoxam (7 mg/plant Platinum 75 SG insecticide, Syngenta Crop Protection LLC, Greensboro, NC, USA) to prevent damage from insect herbivory, and 12 days after the field transplant, all plants were sprayed with fermentation solids, spores, and insecticidal toxins from Bacillus thuringiensis, subsp. kurstaki, strain ABTS-351 (2.6 mL L−1 DiPel Pro dry flowable biological insecticide, Valent Biosciences Corp.) to suppress tobacco hornworm. The field experiment was set up as an incomplete randomized block design with 12 blocks of 6 × 6 plants spaced 30 cm apart (Supplementary Fig. 4). Each block contained four rows of four plants per genotype in north–south (N–S) orientation, surrounded by one border row of WT. WT was present in all blocks (n = 12), whereas the four PSBS overexpression and two psbs knock-down lines were randomly assigned to six blocks (n = 6). The blocks were positioned in a 3 (N–S) × 4 (E–W) rectangle with 75 cm spacing between blocks. The entire experiment was surrounded by two border rows of WT.

Light intensity (LI-190R quantum sensor, LI-COR, Lincoln, NE, USA) and air temperature (Model 109 temperature probe, Campbell Scientific, USA) were measured nearby on the same field site and half-hourly averages were logged using a datalogger (CR1000, Campbell Scientific, USA). Precipitation was measured at two locations close to the field using precipitation gauges (NOAH IV Precipitation Gauge, ETI Instrument Systems Inc., Fort Collins, CO, USA) (Supplementary Fig. 7). Watering to restore field capacity was provided daily when needed through parallel drip irrigation lines with emitters every 30 cm (17 mm PC Drip Line #DL077, The Drip Store, Vista, CA, USA) spanning the whole experiment in E–W orientation and spaced 30 cm apart in N–S direction. To improve soil drainage after watering and precipitation events, two trenches with a depth of approximately 10 cm were dug in N–S direction between the blocks and connected on the south side of the experiment to a 15 cm deep E–W trench. Photosynthesis measurements were performed on the youngest fully expanded leaf 22 days after transplanting. Plants were harvested on July 7, 2016. At final harvest, stem length and the number of leaves were determined, and leaf area was measured with a conveyor-belt scanner (LI-3100C Area meter, LI-COR, Lincoln NE, USA). Leaf, stem, and root fractions were dried to constant weight at 60 °C in a custom-built drying oven equipped with condenser to further dry the recirculated air, after which the dry weights were determined.

Non-photochemical quenching in field-grown plants

Leaf discs were sampled pre-dawn from field-grown plants of psbs-4, psbs-50, PSBS-28, PSBS-34, PSBS-43, PSBS-46, and WT control and stored in darkness in glass vials for up to 4 h until measurement. Humidity in the vials was maintained fully saturated by placing a piece of wet filter paper in each vial. Dark-adapted leaf discs were positioned on a piece of wet filter paper in a chlorophyll fluorescence imager (CFimager, Technologica, Colchester, UK) to determine maximal fluorescence (F m ). Subsequently, leaf discs were exposed to 15 min of 1000 µmol m−2 s−1, after which maximal fluorescence without dark adaptation was determined (F m '). NPQ was then determined according to Eq. 3.

Photosynthetic gas exchange in field

The response of photosynthetic gas exchange to light intensity was measured on the youngest fully expanded leaf of four plants of psbs-4, PSBS-28, PSBS-34, PSBS-43, and WT control in the S–W blocks. Measurements were performed in four complete sets to account for random effects of N–S position of plants, and time of day. Leaves were clamped in the cuvette of an open gas exchange system (LI6400XT, LI-COR, Lincoln, NE, USA) and allowed to reach steady-state gas exchange at saturating light intensity of 2000 µmol m−2 s−1, with block temperature set to 30 °C and [CO 2 ] in the airstream controlled to 400 µmol mol−1 and vapor pressure deficit between air and leaf kept below 1.5 kPa. Subsequently, light intensity was varied from 2,000 to 1,500, 1,000, 800, 600, 400, 300, 200, 170, 140, 110, 80, and 50 µmol m2 s−1. Due to the limited window suitable for measuring gas exchange in field trials, waiting time for steady state was kept between 5 and 10 min for these measurements. When steady state was reached, net assimilation rate, stomatal conductance, and intercellular [CO 2 ] were logged. After gas exchange measurements were performed, leaf absorptance was determined using an integrating sphere (LI1800, LI-COR, Lincoln, NE, USA) connected to a spectrometer (USB-2000, Ocean Optics Inc., Dunedin, FL, USA).

Statistical analysis

All statistical analysis was performed with SAS (version 9.3, SAS Institute Inc., Cary, NC, USA). Data were tested with the Brown–Forsythe test for homogeneity of variance and the Shapiro–Wilk test for normality. One-way analysis of variance was applied to transcription levels, protein expression, gas exchange data, Rubisco content and activation state, stomatal density, and dimension data. Measurement set was included as a random effect in analysis of the field gas exchange data to account for variation caused by N–S plant position and time of day. Biomass, leaf area, and plant height data were analyzed with a linear mixed model accounting for block and genotype effects with Welch–Satterthwaite adjustment of degrees of freedom to account for the different replication rate of WT (PROC MIXED). Significant genotype effects in ANOVA (α = 0.05) were followed by testing of genotype means against WT control (α = 0.05), using Dunnett’s multiple comparison correction. Correlations between Q A redox state with g s and protein levels and with A n /g s were evaluated using Pearson’s correlation coefficient.

Data availability

All relevant data and plant materials are available from the authors upon request. Raw data corresponding to the figures and results described in this manuscript have been deposited at: https://data.mendeley.com/datasets/nsbjps9rkg/draft?a=10508d31-685a-4a62-8f96-cb591c569e97.