Materials and Methods

The study was conducted at the North Willamette Research and Extension Center in Aurora, OR, USA (lat. 45.281009, long. 122.752512) in a heated, double-walled polyethylene greenhouse equipped with automated controls to maintain stable temperature and humidity. The structure provided diffuse light transmission and insulation suitable for cool-season crop production. Wasabi (cv. Daruma) was sourced from a commercial propagator and grown in 10.6-L air-pruning pots (#3 pots; RediRoot, Boring, OR, USA) filled with coconut fiber soilless substrate for greenhouse crops (Rio Coco PCM, Irving, TX, USA).

On 13 Mar 2024, 16 healthy plants were placed on a mini-lysimeter system controlling irrigation based on container weight. Irrigation was triggered at 90% container capacity), defined as the weight after full saturation and 1 h of drainage. The mini-lysimeter control system is discussed in detail in the work of McCauley and Nackley (2022).

Overhead lighting from 1000-W high-pressure sodium lamps were set to provide supplemental morning light at 750 µmol·m⁻²·s⁻¹, operating daily from 8:00 to 11:00 AM, after which natural sunlight provided illumination for the remainder of the day.

Leaf-gas exchange.

A portable photosynthesis system (LI-6800; LI-COR Biosciences, Lincoln, NE, USA) was used to generate light and CO₂ response curves on mature leaves of three different plants (n = 3). Photosynthetic light responses (A/Q) were measured under the following conditions: CO₂ concentration set to 400 ppm, temperature at 21 °C, relative humidity (RH) at 50%, fan speed at 10,000 rpm, and flow rate set to 500 µmol·s⁻¹. Readings were taken at 2000, 1500, 1000, 800, 600, 500, 300, 200, 150, 50, and 0 photosynthetic photon flux density (PPFD).

Photosynthetic CO₂ response (A/C_i) were measured under the following conditions: incident light (Q_in) was maintained at 650 µmol·m⁻², with a temperature of 21 °C, RH at 50%, fan speed at 10,000 rpm, and flow rate set to 600 µmol·s⁻¹. Measurements were taken at CO₂ concentrations of 400, 300, 200, 100, 50, 0, 400, 600, 800, 1000, 1200, 1600, and 2000 ppm over a period of ∼45 min.

CO₂ response data were analyzed using the Farquhar–von Caemmerer–Berry (FvCB) model (Farquhar et al. 1980) adjusted for temperature and CO₂ (Bernacchi et al. 2001). Parameters included V_{c max}, J_max, R_d, and triose phosphate utilization (TPU) (Sharkey et al. 2016). Parameters were estimated using nonlinear least squares in MATLAB constrained within biological ranges and evaluated with linear modeling (Supplemental Fig. 1).

Starting on 18 Mar 2024, irrigation was stopped, and the pots were allowed to dry down. During a 4-day dry-down, leaf gas-exchange parameters (g_sw, VPD_leaf, and Q_amb) (Table 1) were measured from each plant (n = 16) every 60 to 90 min starting at 9:00 AM until around 4:00 PM with a portable porometer–fluorometer (LI-600; LI-COR Biosciences). Attention was focused on sampling the newest, fully developed mature leaves.

Table 1. Environmental data recorded on the 4 days when soil moisture content and transpiration measurements were taken.

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Results and Discussion

The automatic misting and irrigation systems in the greenhouse were used to effectively mimic natural conditions of the mountain stream beds with abundant moisture and low heat stress. During the initial phase, when moisture was abundant, leaf gas exchange revealed a maximum assimilation rate (A_max) of 11.08 ± 0.42 µmol·m⁻²·s⁻¹ at 828 µmol·m⁻²·s⁻¹ Q_amb (Supplemental Fig. 1). Model fitting showed a strong correlation between light levels and net assimilation (R² = 0.906), with adequate PPFD setting for greenhouse growers to target between 500 and 800 µmol·m⁻²·s⁻¹. The A/C_i curve (Supplemental Fig. 1B) showed an A_max of 22.84 ± 1.82 µmol·m⁻²·s⁻¹ at 1600 µmol·mol⁻¹ CO₂, indicating a strong response to CO₂ but with diminishing returns at higher CO₂ concentrations. Key parameters such as V_{c max} (74 μmol·m⁻²·s⁻¹ at 21 °C) and J_max (108 μmol·m⁻²·s⁻¹ at 21 °C) suggest that photosynthesis in wasabi is constrained by both Rubisco activity and RuBP regeneration at lower CO₂, with a triose phosphate utilization (TPU) limitation at higher concentrations. The FvCB model fit strongly (R² = 0.959). The higher V_cmax and J_max values suggest that wasabi has the potential to benefit from elevated CO₂ conditions, making greenhouse cultivation with CO₂ enrichment targeting 1200 ppm CO₂ a viable option for future cultivation.

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During the dry-down period, when irrigation was limited, the drying substrate caused rapid reductions in stomatal conductance (g_sw), indicating a sensitivity to dry soils. While g_sw was positively correlated with both Q_amb and VPD_leaf, field capacity (FC) emerged as the dominant control. For example, at 95% FC, g_sw remained robust (∼3 to 4 µmol H₂O m⁻²·s⁻¹) even as VPD_leaf approached 2.0 kPa (Fig. 1). In contrast, at 82% FC, g_sw remained low (<1 µmol H₂O m⁻²·s⁻¹) across all VPD_leaf and ambient light (Q_amb) conditions. Plants at 95% and 90% FC showed a clear positive response to increasing light, with peak g_sw observed around 800 µmol·m⁻²·s⁻¹. However, plants at 86% and 82% FC displayed minimal g_sw response to changing light levels, indicating that light alone could not overcome the limitations imposed by lower soil moisture. Our work is among the very few studies of wasabi photosynthesis, with essentially no direct comparisons available. A couple of titles from the early 2000s suggest that photosynthesis work was conducted in Korea, but these studies were published in Korean in reports that are not readily accessible outside that region. A more recent publicly available study examined the effects of light quality on wasabi growth (Ruamrungsri et al. 2025). However, according to our findings, their research was conducted at light intensities (35, 60, 90, and 140 µmol·m⁻²·s⁻¹ PPFD) that were suboptimal. These are unusually low light intensities for any plant. We identified an optimal target of 800 ± 50 µmol·m⁻²·s⁻¹ PPFD, which supported assimilation rates of ∼10 µmol·m⁻²·s⁻¹, increasing to >20 µmol·m⁻²·s⁻¹ with CO₂ enrichment (Supplemental Fig. 1). By comparison, their reported assimilation rates of ∼2 µmol·m⁻²·s⁻¹ (Ruamrungsri et al. 2025) were consistent with our measurements at ∼100 µmol·m⁻²·s⁻¹ PPFD. Given the scarcity of wasabi research, indirect comparisons to other Brassicaceae root crops provide a useful benchmark. For example, red radish (Raphanus sativus L.) A_max was between 15 and 18 µmol·m⁻²·s⁻¹ and was sensitive to irrigation deficit (Alsadon et al. 2023). These findings underscore the critical role of soil moisture in maintaining stomatal function and suggest that irrigation strategies for wasabi should prioritize keeping FC above 90% to sustain physiological activity under fluctuating environmental conditions.