Photosystem II Subunit S overexpression increases the efficiency of water use in a field-grown crop

Abstract

Insufficient water availability for crop production is a mounting barrier to achieving the 70% increase in food production that will be needed by 2050. One solution is to develop crops that require less water per unit mass of production. Water vapor transpires from leaves through stomata, which also facilitate the influx of CO₂ during photosynthetic assimilation. Here, we hypothesize that Photosystem II Subunit S (PsbS) expression affects a chloroplast-derived signal for stomatal opening in response to light, which can be used to improve water-use efficiency. Transgenic tobacco plants with a range of PsbS expression, from undetectable to 3.7 times wild-type are generated. Plants with increased PsbS expression show less stomatal opening in response to light, resulting in a 25% reduction in water loss per CO₂ assimilated under field conditions. Since the role of PsbS is universal across higher plants, this manipulation should be effective across all crops.

Fig. 1

Photosynthesis and water-use efficiency in Nicotiana tabacum plants with modified PsbS levels. a PsbS mRNA levels normalized to actin and tubulin sampled from fully expanded leaves of psbs-4, PSBS-28, PSBS-43 and wild-type (WT) N. tabacum plants. b PsbS protein levels normalized to the large subunit of oxygen-evolving complex of photosystem II (PsbO), determined from densitometry on immunoblots. c Representative immunoblot for PsbS and PsbO. d Net CO₂ fixation rate (A_n), e NPQ, f quinone A (Q_A) redox state, and g stomatal conductance (g_s) as a function of incident light intensity in fully expanded leaves. h Linear correlation between Q_A redox state and g_s. Broken lines indicate measurements at highest light intensity. i Linear correlation between PsbS protein levels and intrinsic water-use efficiency (A_n/g_s) at light intensity above 600 μmol m⁻² s⁻¹. Asterisks/lines show significant differences from WT (black for silencing, red for overexpressing lines; Dunnett’s two-way test; α = 0.05). Error bars indicate s.e.m. (n = from 6 to 10 biological replicates)

Fig. 2

PsbS expression and photoprotection in Nicotiana tabacum plants grown under field conditions. a–d PsbS mRNA levels and e–h PsbS protein levels in several tobacco genotypes with modified PsbS expression levels as well as wild-type (WT) tobacco at four time points during the field experiment (DAE = days after emergence). i Representative levels of NPQ for each genotype determined on leaf discs. All genotype means were significantly different from WT (Dunnett’s two-way test; P ≤ 0.008 for mRNA; P ≤ 0.001 for protein). Error bars indicate s.e.m. (n = 4 biological replicates)

Fig. 3

Photosynthetic water-use efficiency and productivity of field-grown Nicotiana tabacum plants with modified PsbS levels. a Net CO₂ fixation rate (A_n), b stomatal conductance (g_s), c intrinsic water-use efficiency (A_n/g_s) in tobacco genotypes with modified PsbS expression levels relative to wild-type tobacco. d Linear correlation between PsbS protein levels and A_n/g_s at light intensity above 300 μmol m⁻² s⁻¹. e Total dry weight, f Leaf area, and g plant height. Error bars indicate s.e.m. (a–d N = 4 biological replicates; e–g n = 6 blocks for transgenic and n = 12 blocks for WT), and asterisks indicate significant differences between transgenic lines and WT (Dunnett’s two-way test; α = 0.05), P-values indicate significance of line effect in ANOVA (a–c and e–g) or significance of regression (d)

Fig. 4

Interactions between light and CO₂ control over stomatal movements. a Schematic representation of processes in the chloroplast thylakoid membrane, Calvin–Benson cycle in the chloroplast stroma, and at the interchange between intercellular airspace and atmosphere through the stomatal pore. Indicated are where the CO₂ signal (C_i signal) and the proposed Q_A-redox signal originate. b Chain of events showing the direct effects on the stomatal control signals from C_i and Q_A redox state of the manipulation of PsbS expression in the current work and several previously published manipulations, and the subsequent direction of change in stomatal conductance (g_s). Superscripted numbers indicate corresponding literature references. Abbreviations: 3PGA – 3-Phosphoglycerate; ADP – adenosine diphosphate; ATP – adenosine triphosphate; Cyt b₆f – Cytochrome b₆f; DCMU – (3-(3,4-dichlorophenyl)-1,1-dimethylurea); FBP aldolase – Fructose-bisphosphate aldolase; Fd – Ferredoxin; FNR – Ferredoxin NADP(+) reductase; G3P – Glyceraldehyde-3-phosphate; GAPDH – Glyceraldehyde 3-phosphate dehydrogenase; LHCI or II – light-harvesting complex I or II; NADP+ / NADPH – nicotinamide adenine dinucleotide phosphate (oxidized/reduced); NPQ – non-photochemical quenching; OEC – Oxygen evolving complex; PC – Plastocyanin; Pheo – pheophytin; PRK – Phosphoribulokinase; PsbO – subunit of the oxygen evolving complex (OEC); PsbS – Photosystem II subunit S; PSI or II – photosystem I or II; Q_A – Plastoquinone A; Q_B – Plastoquinone B; Rubisco – Ribulose-1,5-bisphosphate carboxylase-oxygenase; RuBP – Ribulose 1,5-bisphosphate, SBPase – Sedoheptulose-bisphosphatase