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Article

Cultivar-Specific Responses of Spinach to Root-Zone Cooling in Hydroponic Systems in a Greenhouse Under Warm Climates

by
Md Noor E Azam Khan
,
Joseph Masabni
and
Genhua Niu
*
Texas A&M AgriLife Research and Extension Center, Dallas, TX 75252, USA
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(9), 3925; https://doi.org/10.3390/su17093925
Submission received: 12 March 2025 / Revised: 19 April 2025 / Accepted: 25 April 2025 / Published: 27 April 2025
(This article belongs to the Special Issue Sustainable Agriculture with Innovative Technology and Equipment: Towards a Low-Carbon Era)
Browse Figures
Versions Notes

Abstract

:
Growing spinach year-round via greenhouse hydroponics in warm climates can be challenging because of the intolerance of many spinach cultivars to heat. Root-zone cooling in hydroponic systems in warm climates may be a promising cooling method to alleviate heat stress; however, its effectiveness is still unknown in spinach plants. This study aimed to investigate the impact of root-zone cooling on the growth and physiological responses of four spinach cultivars (‘Lakeside’, ‘Hammerhead’, ‘Mandolin’, and ‘SV2157’) grown in deep water culture hydroponic systems in a greenhouse during the summer season in two growing cycles. The experiment consisted of the following three root-zone temperatures (RZTs): Control (ambient water temperature), RZT24 (24 °C), and RZT21 (21 °C). Among the four cultivars, ‘SV2157’ performed equally regardless of the treatment, demonstrating superior heat tolerance versus the other three cultivars. ‘Mandolin’ exhibited the greatest benefit from root-zone cooling, with increases in shoot dry weights of 87% and 94% under RZT24 and RZT21, respectively, compared to those under control treatment. Additionally, total leaf areas significantly increased under the two root-zone cooling treatments. ‘Lakeside’ and ‘Hammerhead’ generally benefited from root-zone cooling, although the magnitude of growth increases was small or statistically insignificant. However, ‘Lakeside’ and ‘Hammerhead’ were highly responsive to lower ambient air temperatures, as evidenced by increases of 121% and 90%, respectively, in shoot fresh weights across the treatments in Cycle 2 (average air temperature of 24.7 °C) compared to those in Cycle 1 (29.3 °C). Physiological responses to root-zone cooling varied among cultivars, with ‘SV2157’ exhibiting the highest chlorophyll, carotenoid, and anthocyanin levels. Higher total phenolic contents under control treatment in Cycle 1 in all three cultivars except for ‘SV2157’ suggested greater reactive oxygen species production, indicating oxidative stress. Root-zone cooling reduced oxidative stress indicators, including mortality (%), hydrogen peroxide content, and malondialdehyde content, and minimized cell leakage. Based on plant growth, physiological and biochemical traits, and electricity consumption, cooling the root zone to 24 °C rather than 21 °C is recommended for hot summers with high air temperatures.

1. Introduction

Food security and sustainable production systems are increasingly prioritized in today’s rapidly growing world. The production of leafy greens plays a crucial role in addressing food security due to their widespread consumption and demand. The global leafy greens market was valued at USD 83.95 billion in 2024, with forecasts predicting an increase to USD 88.40 billion in 2025 and a further increase to USD 140.74 billion by 2034 [1]. This growing demand compels growers to choose the best production methods based on consumer preferences. Among the various leafy greens, spinach (Spinacia oleracea L.) is highly popular, being valued for its rich contents of essential vitamins (A, C, K), minerals (iron, calcium), and other beneficial phytochemicals [2,3]. Additionally, fresh or raw spinach contains compounds such as (Z)-3-hexenal and methanethiol, which contribute to its unique aroma, enhancing its appeal to consumers [4].
Hydroponic crop production systems are well suited for leafy greens production, and these systems are widely used in greenhouses and indoor farms. Research has shown that hydroponic lettuce production can yield up to 20 times more per acre than traditional soil-based cultivation [5]. Spinach is more heat sensitive than lettuce; therefore, growing spinach hydroponically in a greenhouse in warm climates is challenging in the summer season [6]. Regulation of the air temperature of an entire greenhouse through heating or cooling leads to high energy costs, and maintaining ideal air temperature conditions is difficult during the hot summer when outside air temperatures are high [7]. On the other hand, spinach grows best below 23 °C and experiences metabolic stress above 30 °C [8,9]. In the southern states of the USA, greenhouse air temperature can easily reach above 35 °C even with regular cooling methods like the combination of shading and evaporative cooling. Under such high-temperature conditions, growing spinach in a greenhouse hydroponic system becomes difficult, and maintaining the air temperature of a greenhouse at an ideal level for spinach in hot and humid summers is impossible using evaporative cooling and shading. Notably, an increase in temperature from 25 to 35 °C leads to a 24.68% reduction in the fresh leaf biomass of spinach [10]. High temperatures reduce photosynthetic efficiency, leading to decreased growth and yield. The activity of photosystem II (PSII) is particularly affected, resulting in reduced quantum yields and increased energy dissipation [11]. Moreover, high air temperatures increase the accumulation of reactive oxygen species (ROS) and associated enzymes, causing cellular membrane damage and oxidative stress, which can lead to cell death [12]. Therefore, to ensure year-round production and overcome seasonal challenges, practical solutions are needed to alleviate heat stress in hydroponic spinach production.
One promising solution in greenhouse hydroponics is cooling the root-zone temperature by placing a chiller in the nutrient reservoir, which directly influences plant physiology and growth [13]. Cooling the root-zone temperature under high ambient greenhouse air temperature may alleviate heat stress, especially for cool-season crops. For instance, maintaining a root-zone temperature of 21.1 °C increased lettuce shoot fresh weight and nutrient uptake compared to ambient conditions [14]. Similarly, cooling the root zone using a cooling tube in soil-grown spinach led to increased shoot fresh weight and improved growth [15]. Other crops, including cucumber, tomato, and salad rocket, also showed improved growth with root-zone cooling [16,17,18,19]. However, there is still limited information regarding cultivar-specific responses in spinach grown hydroponically.
Root-zone cooling may impact the physiological and biochemical responses of plants, particularly under high-temperature stress. This is because high temperatures can stimulate ROS production in plants, leading to the accumulation of bioactive compounds such as total phenolics, flavonoids, ascorbic acid, and other antioxidants [20,21]. These compounds neutralize ROS, enhancing the plants’ resilience to stress [22]. Regulating the root-zone temperature also influences root membrane fluidity and aquaporin activity, affecting water and nutrient uptake while triggering stress response pathways mediated by calcium channels, heat shock proteins, and ROS-scavenging mechanisms [23,24,25]. These processes are crucial for maintaining optimal growth and quality in heat-stressed crops. In addition, high temperatures can reduce dissolved oxygen levels in water, leading to suppressed root nutrient update and growth. Cooling the root zone in hydroponic systems can support stable nutrient uptake, reduce root disease risks, and create favorable conditions for plant growth under high temperatures [26].
Our previous study revealed promising results for root-zone cooling to 24 °C in several leafy greens, including the two spinach cultivars ‘Mandolin’ and ‘Seaside’ [6]. The objective of this study was to investigate the impact of root-zone cooling to 24 °C and 21 °C on four representative spinach cultivars grown in hydroponic systems in the summer under greenhouse conditions. The goal of this research was to validate root-zone cooling as a feasible, energy-efficient strategy to enhance spinach yields and quality, supporting sustainable, year-round hydroponic spinach production. We chose four varieties—’SV2157’, Hammerhead’, ‘Mandolin’, and ‘Lakeside’—in this study. ‘Mandolin’ was more heat tolerant than ‘Seaside’, although the plants did not grow out [6]. ‘Lakeside’ is a similar variety to ‘Seaside’, and ‘SV2157’ is a potentially heat-tolerant cultivar, which was recommended by the seed company described below and has dark, glossy leaves with good yield potential. ‘Hammerhead’ has medium-green, upright spinach leaves and showed good yield potential in our previous hydroponic trial (unpublished data).

2. Materials and Methods

2.1. Seedling Propagation

Four cultivars of spinach (Spinacia oleracea) (‘Lakeside’, ‘Hammerhead’, ‘Mandolin’, and ‘SV2157’) were selected for this experiment. Seeds of ‘Lakeside’, ‘Mandolin’, and ‘SV2157’ were purchased from Osborne Quality Seeds (Mount Vernon, WA 98273, USA), and ‘Hammerhead’ seeds were sourced from Johnny’s Selected Seeds (Winslow, ME 04901, USA). Before sowing, the seeds were soaked in distilled water for 12 h to soften the seed coat to aid in germination. The rockwool was rinsed with tap water, and at least two seeds were sown per rockwool cube. Rockwool sheets with 200 cubes were placed in a nursery tray (51 cm length × 25.5 cm width × 6.4 cm depth). These trays were transferred into a reach-in growth chamber with a temperature setpoint of 18 °C for the first four days. Trays were covered with a humidity dome to maintain moisture during germination. Four days after sowing, the domes were removed, and the trays were transferred into a germination rack where three LED light bars (PPFD of 100 µmol/m2/s for the first 5 days and then increased to 200 µmol/m2/s) were turned on per shelf for 12 h/day. Seedlings were sub-irrigated with a nutrient solution (electrical conductivity [EC] of 1.0 mS/cm, pH of 6.1) when the rockwool cubes’ surfaces dried out. The propagation area was maintained at room temperature (approximately 22–25 °C). When seedlings reached the 2–3 true leaf stage, or 19 days after sowing, the seedlings were transplanted into nine deep water culture (DWC) hydroponic systems.

2.2. Experimental Design and Treatments

The experiment was carried out in a glass greenhouse at the Texas A&M AgriLife Research and Extension Center in Dallas, TX 75252, USA (32.77° N, 96.80° W) from July to September 2024. The experiment was repeated two times (two cycles). The experiment had a two factorial randomized complete block design (RCBD) with three root-zone temperature treatments (RZT) (without cooling or control; 23.9 °C ≈ 24 °C, RZT24; and 21.1 °C ≈ 21 °C, RZT21) and four cultivars (‘Lakeside’, ‘Hammerhead’, ‘Mandolin’, and ‘SV2157’). A total of nine DWC systems (three systems or replicates per treatment) were used (Supplementary Figure S1). A floating raft holding 36 plants (nine plants/cultivar) was used per DWC. One DWC system was considered one experimental unit. Each DWC, with dimensions of 1.65 m × 0.75 m × 0.24 m, was filled with 100 L of the nutrient solution. The nutrient solution was made following a modified formula for leafy greens (in mg/L: N 150, P 30, K 193, Ca 135, Mg 40, S 52, Fe 2.00, B 0.31, Mn 0.21, Zn 0.11, Cu 0.05, Mo 0.05 ppm) according to Hooks et al. [6]. For RZT24 and RZT21, each DWC was equipped with an ActiveAQUA AA hydroponic chiller and a submersible water pump to maintain the root-zone temperature settings. All DWC systems were provided with two sets of air stones to aerate the system to provide sufficient dissolved oxygen (DO). The solution temperature was stabilized 24 h before transplanting. Daily EC and pH were measured by using a Bluelab combo meter (HI98129, Hanna instruments, Smithfield, RI 02917, USA), and DO was measured with a YSI Pro Solo DO meter. In addition, daily electricity consumption by chillers was recorded using a SURAIELEC energy watt meter. Table 1 and Figure 1 illustrate the other basic environmental conditions during the two cycles of the experiment.

2.3. Data Collection

2.3.1. Growth and Morphology

Plants were harvested after 22 days of treatment. The following growth and morphological parameters were collected: shoot fresh weight (FW) and shoot dry weight (DW), root fresh weight and root dry weight, total leaf area (LA), third leaf length (LL), third leaf width (LW), leaf number (LN), stem length (SL), and stem diameter (SD). The day before harvesting, LL and LW were recorded using a measuring scale. Plants were cut at the propagation substrate level and weighed immediately for shoot FW and root FW before being placed in paper bags and dried in a drying oven (Thermo Fisher Scientific, Waltham, MA, USA) at 80 °C for 48 h to measure shoot DW and root DW. Leaves were separated from the basal stem, and LA was measured using an LI-3100C Leaf Area Meter (LI-COR, Lincoln, NE, USA). After leaf separation, SL was assessed with a measuring scale and SD with a Vernier scale.

2.3.2. Plant Physiological Responses and Phytochemical and Biochemical Analyses

Chlorophyll Fluorescence and Pigments

The maximum quantum efficiency, Fv/Fm, was determined during midday by using an OS5p+ chlorophyll fluorimeter (OPTI-SCIENCES, Hudson, NH 03051, USA). One representative mature leaf per plant was chosen to measure Fv/Fm following 15 min of dark adaptation. A total of four plants per cultivar per treatment were measured. Plant pigments, namely, total chlorophyll (Chla + Chlb), carotenoids, and anthocyanin were measured by following the specific protocols developed by Wellburn [27] with slight modification. Immediately after harvesting, all plant tissues were ground with liquid nitrogen to avoid any loss of phytochemicals and were stored in a −80 °C freezer until measurement. Fresh plant tissue (50 mg) was taken and mixed with 1.5 mL of high-grade methanol in 2 mL brown tubes (to ensure protection from light exposure) for each sample. Then, the samples were kept in complete darkness at 4 °C for 24 h. The samples were vigorously vortexed under low light conditions and then centrifuged at 10,000 rpm (Eppendorf 5417R microcentrifuge, Hamburg, Germany) for 5 min at 10 °C, resulting in a green supernatant. For absorbance measurements, 1 mL of each sample was transferred to a 1 cm path-length cuvette, and readings were taken using a spectrophotometer (Genesys 10S UV–VIS, Thermo Scientific, Madison, WI, USA) at 665.2 nm (chlorophyll a), 652.4 nm (chlorophyll b), and 470 nm (carotenoids). Pigment concentrations were calculated using the following Wellburn [27] equations:
Chl a (µg/mL) = 16.72 × A665.2 − 9.16 × A652.4
Chl b (µg/mL) = 34.09 × A652.4 − 15.28 × A665.2
Car (µg/mL) = [1000 × A470 − 1.63 × Chl a − 104.96 × Chl b]/221
Anthocyanin was measured following the method of Dou et al. [28]. To start, 200 mg of the ground sample was transferred into a 2 mL microtube, followed by the addition of 1.5 mL of 1% acidified methanol, which was prepared by adding 10 mL of HCl to 990 mL of methanol. The mixture was thoroughly vortexed and incubated in darkness at 4 °C for 12–15 h. After overnight extraction, the samples were centrifuged at 10,000 rpm (Eppendorf 5417R microcentrifuge, Hamburg, Germany) for 15 min, and 1 mL of the supernatant was collected for subsequent phytochemical analysis. The absorbance of the extracts was measured at 520 nm, using 1% acidified methanol as a blank control. The results were expressed as mg cyanidin-3-glucoside equivalents (CE) and calculated from following equation:
Anthocyanin (mg/g FW) = (V × M × A)/(ε × m)
where V is the volume of extraction liquid (mL), M is the molecular weight of cyaniding-3-glucoside, 449.2 g/mole, A is the absorbance at 520 nm, ε is the molar extinction coefficient, 29,600, and m is the weight of the sample.

Phytochemicals

Total phenolic and flavonoid contents were quantified by following the procedures used by Chew et al. [29] and Tan et al. [30], respectively, with slight modifications. All samples were kept frozen at −80 °C before extraction. For each sample, a mid-sized leaf was selected, ground into a powder using liquid nitrogen with a mortar and pestle, and weighed approximately 200 mg. The ground sample was transferred to a 2 mL microtube, and 1.5 mL of 1% acidified methanol was added. The mixture was vortexed thoroughly and incubated overnight (12–15 h) in darkness at 4 °C. After incubation, the samples were centrifuged at 10,000 rpm (Eppendorf 5417R microcentrifuge, Hamburg, Germany) for 15 min, and 1 mL of supernatant was collected for phytochemical analysis. For the total phenolic content, a 20 µL aliquot of the extract was transferred to a 96-well microplate, followed by the addition of 40 µL of 1/10 dilution Folin–Ciocalteu reagent. After 6 min, 160 µL of 7.5% sodium carbonate (Na2CO3) was added, and the mixture was shaken for 30 s, followed by incubation at room temperature for 2 h. Absorbance was measured at 725 nm using a plate reader (ELx800, BioTek, Winooski, VT, USA). For the total flavonoid content assay, 20 µL of the extract was added to a microplate with 85 µL of distilled water and 5 µL of 5% sodium nitrite (NaNO2). After 6 min, 10 µL of 10% aluminum chloride hexahydrate (AlCl3·6H2O) was added, followed by another 5 min of reaction time. Then, 35 µL of 1 M sodium hydroxide (NaOH) and 20 µL of distilled water were added, the mixture was shaken vigorously, and absorbance was measured at 520 nm using the ELx800 microplate. Each assay was accompanied by blank and standard curve preparations. The results were expressed as mg gallic acid equivalents (GAE) per gram of fresh weight for the total phenolic content (TPC) and mg catechin equivalents (CE) per gram of fresh weight for the total flavonoid content.

2.3.3. Stress Indicators

Mortality (%) was considered a stress indicator influenced by the growth conditions, specifically, high air temperatures. Throughout each production cycle, dead plants (without any visible green tissues) were counted, and final mortality (%) was calculated by the following equation:
Mortality (%) = (Number of dead plants before harvesting)/(Total number of plants) × 100
The content of hydrogen peroxide (H2O2) in spinach leaves was measured following the protocol of Jessup et al. [31]. Briefly, approximately 0.1 g of sample was ground into 2 mL of 0.1% trichloroacetic acid (TCA). Then, samples were centrifuged at 12,000 rpm (Eppendorf 5417R microcentrifuge, Hamburg, Germany) for 15 min at 4 °C. Next, potassium iodide (1 mL) and potassium phosphate buffer pH 7.0 (0.5 mL) were added to the supernatant (0.5 mL). The mixture was vortexed, and then absorbance was recorded at 390 nm using the 10S UV–VIS spectrophotometer. The amount of H2O2 was calculated using a standard curve and expressed in μmol/g FW.
Lipid peroxidation can be recognized from malondialdehyde (MDA) in plant tissue. The measurement method was adapted from Heath and Packer [32]. First, a leaf tissue sample (0.1 g) was homogenized using 5% TCA. The homogenates were centrifuged for 15 min at 12,000 rpm (Eppendorf 5417R microcentrifuge, Hamburg, Germany). Then, the supernatant (0.5 mL) was added to 0.5% thiobarbituric acid (1 mL). The mixture was incubated in a water bath at 95 °C for 30 min. Subsequently, the samples were cooled under room temperature and recentrifuged at 7500 rpm (Eppendorf 5417R microcentrifuge, Hamburg, Germany) for 5 min. The absorbance was recorded at 532 and 600 nm using the 10S UV–VIS spectrophotometer. For the blank, 5% TCA was used. The final amount of MDA in leaf tissues was calculated by using the following equation:
MDA (nmol/g fresh weight) = {(Absorbance at 532 nm − Absorbance at 600 nm)/(ε × W)} × 106
where W is the weight of fresh tissue (g) and ε is 155 m/M/cm (extinction coefficient of MDA).
Membrane integrity was measured based on the percentage of cell or solute leakage. This was estimated according to Vinodh and Kannan [33] with slight modifications. A leaf tissue sample (0.5 g) and 20 mL of water were mixed together and kept for two hours. Later, the absorbance was read at 273 nm. Then, the same mixture was boiled for 10 min, and the absorbance was measured again. The percentage of solute leakage was calculated using the following formula:
% Cell leakage = {(Final absorbance at 273 nm − Initial absorbance at 273 nm)/(Final absorbance at 273 nm)} × 100
For all the biochemical analyses used for the stress evaluation, plant tissue was previously ground with liquid nitrogen and stored at −80 °C to preserve the volatile and labile compounds during extraction [34].

2.4. Statistical Analyses

A two-way analysis of variance (ANOVA) was carried out to determine the main effects of root-zone temperature and cultivars. The means were compared, and an honestly significant difference (HSD) test with a 5% significance level was conducted when the main effect was significant. When there was no interaction between root-zone temperature and cultivar, the data were pooled. Since the greenhouse environmental conditions between the two cycles were significantly different due to differences in weather conditions, as shown in Figure 1, statistical analyses were conducted independently for the two cycles, and the data were presented separately. These analyses were conducted in RStudio (Version 4.4.2). Other graphical representations were created using Microsoft Excel 365.

3. Results

Root-zone temperature affected all growth parameters and stress indicators in Cycle 1 (Table 2). There were significant interactive effects between RZT and cultivar (CV) on all growth parameters, the total phenolic content (TPC), and stress indicators, indicating variability in cultivar responses to root-zone cooling. No interactive effects between RZT and cultivar were observed on morphological parameters (Table 3), the maximum quantum yield (Fv/Fm), or total chlorophyll, total carotenoid, anthocyanin, and total flavonoid contents (TFCs) in Cycle 1. However, only anthocyanin was significantly affected by RZT in Cycle 2 (Supplementary Table S1). For electricity consumption, the daily average electricity consumption in Cycle 1 was 1.23 kWh under RZT24 and 2.67 kWh under RZT21 (Table 1). In Cycle 2, the daily average electricity consumption levels were 0.83 kWh and 1.84 kWh, respectively, under RZT24 and RZT21. The average air temperature differed significantly between the two experimental cycles and was recorded as 29.3 °C in Cycle 1 and as 24.7 °C in Cycle 2. Similarly, the average solution temperature under the control treatment also varied across cycles, with temperatures of 28.0 °C in Cycle 1 and 23.9 °C in Cycle 2. Notably, Figure 1B shows examples from the 14th day of both Cycle 1 and Cycle 2, showing the typical 24 h fluctuations of both air and root-zone temperatures during this study. The air and solution temperatures in Cycle 2 decreased significantly compared to those in Cycle 1.

3.1. Growth and Morphological Parameters

In Cycle 1, root-zone cooling significantly increased shoot FW, shoot DW, root DW, and total leaf area only in ‘Mandolin’ (Figure 2A,C). In ‘Mandolin’, shoot FW increased by approximately 150% and 171% under RZT24 and RZT21, respectively, compared to that with control treatment. Likewise, shoot DW increased by 87% and 94% under RZT24 and RZT21, respectively, compared to that in the control. Root DW increased by approximately 89% and 80% under RZT24 and RZT21, respectively, compared to that under control treatment (Figure 2E). Moreover, the LA in ‘Mandolin’ increased by approximately 126% and 164% under RZT24 and RZT21, respectively, relative to that in the control. For the other three cultivars, there were no statistical differences among the treatments for all growth parameters. However, there was a tendency for root-zone cooling to increase shoot growth in ‘Lakeside’ and ‘Hammerhead’, as evidenced by the numerical increases in both shoot FW and DW under RZT24 and RZT21 in comparison with observations in the control. In ‘SV2157’, shoot growth was similar among the treatments and had a relatively high yield, indicating that it was heat tolerant.
Conversely, in Cycle 2, none of the growth parameters were significantly affected by any factors (RZT, CV, and RZT × CV). However, both ‘Lakeside’ and ‘Hammerhead’ exhibited improved growth characteristics in Cycle 2 (Figure 2B,D,F,H) compared to Cycle 1. For instance, the shoot FW of ‘Lakeside’ in Cycle 2 increased by approximately 160%, 137%, and 66% in the control, RZT24, and RZT21 groups, respectively, compared to that in Cycle 1. Additionally, in ‘Lakeside’, shoot DW, root DW, and LA showed average increases of 68%, 32%, and 92% across root-zone temperatures compared to those in Cycle 1. In ‘Hammerhead’, shoot FW, shoot DW, root DW, and LA in Cycle 2 increased by approximately 90%, 85%, 47%, and 94%, respectively, across the RZTs compared to those in Cycle 1.
In Cycle 1, morphological parameters exhibited cultivar-specific variations, except for SL (Table 3 and Supplementary Figure S2). There were no interactive effects between RZT and cultivar on any morphological parameters. Generally, root-zone cooling increased leaf length (LL), leaf width (LW), and stem diameter (LD), indicating a positive effect of promoting growth. Among the cultivars, ‘Lakeside’ had the lowest LL (11.45 cm) and LW (6.65 cm) compared to the other three cultivars. Leaf number and stem diameter were lower in ‘Lakeside’ and ‘Hammerhead’ than in the other two cultivars. In Cycle 2, no significant differences were observed across the factors (Supplementary Table S1).

3.2. Physiological Attributes

Root-zone temperature did not affect Fv/Fm; however, there were cultivar-specific variations (Figure 3A), with ‘Hammerhead’ exhibiting the lowest value (0.63), while no statistical differences were found among the other three cultivars.
Significant differences among cultivars were also observed in leaf pigments, including total chlorophyll, anthocyanin, and carotenoids (Figure 3B–D). ‘SV2157’ consistently exhibited the highest pigment levels, with total chlorophyll, anthocyanin, and carotenoids averaging 52%, 14%, and 46% higher, respectively, than those in the other cultivars. RZT treatment resulted in only slight differences in anthocyanin accumulation (Figure 3C).
Total phenolic content (TPC) was significantly affected by all experimental factors, and there was a significant interaction of RZT × CV (Table 2). Root-zone cooling reduced TPC levels in all cultivars except ‘SV2157’. Compared to the control, ‘Lakeside’ showed 71% and 36% reductions in TPC under RZT24 and RZT21, respectively (Figure 3E). Similarly, TPCs in ‘Hammerhead’ and ‘Mandolin’ decreased more under RZT24 (by approximately 47% and 43%, respectively) than under RZT21. In contrast, total flavonoid content (TFC) remained unaffected by RZT, CV, and their interaction (Table 2).
In Cycle 2, no significant differences were observed between any physiological attributes across all factors, except for anthocyanin in response to RZT (Supplementary Table S1).

3.3. Stress Indicators

All stress parameters were significantly affected by all factors in Cycle 1 (Table 2). Without root-zone cooling, most cultivars exhibited significant stress (Figure 4). However, in Cycle 2, all the factors were nonsignificant (Supplementary Table S1); hence, the results from Cycle 2 are not presented in this section.
Among the four cultivars, ‘Hammerhead’ exhibited the highest mortality (14.81%) without root-zone cooling (control). ‘Mandolin’ had a 3.7% mortality rate only under control treatment (Figure 4A). Root-zone cooling reduced mortality in ‘Hammerhead’ and ‘Mandolin’ compared to the control treatment. In contrast, both ‘Lakeside’ and ‘SV2157’ were not significantly affected by the root-zone temperatures.
Root-zone cooling significantly reduced H2O2 accumulation, MDA contents, and cell leakage (%) in all cultivars except for ‘SV2157’, which showed no significant variation across treatments (Figure 4B–D). In ‘Hammerhead’, H2O2 levels decreased by approximately 59% under RZT24 and 63% under RZT21 compared to those under control treatment. ‘Mandolin’ also exhibited a notable reduction of 59% under RZT24 relative to the control. MDA levels followed a similar trend to H2O2, except in ‘Hammerhead’, where they decreased by approximately 38% and 35% under RZT24 and RZT21, respectively, compared to those with control treatment. ‘Hammerhead’ and ‘Mandolin’ showed average reductions of 46% and 36% under RZT24 and RZT21, respectively, compared to the control. In ‘Lakeside’, MDA levels also decreased by approximately 55% and 52% under RZT24 and RZT21, respectively, in contrast to those under control treatment. Cell leakage (%) exhibited a substantial decline under root-zone cooling in ‘Lakeside’, ‘Hammerhead’, and ‘Mandolin’. In ‘Hammerhead’, cell leakage decreased by approximately 69% and 81% under RZT24 and RZT21, respectively, compared to that with control treatment. In ‘Mandolin’, cell leakage decreased by approximately 80% and 74% under RZT24 and RZT21, respectively, relative to that under control treatment. A notable reduction was also observed in ‘Lakeside’ under RZT24 (6.4%) and RZT21 (5.7%) compared to the control (24.1%).

4. Discussion

4.1. Root-Zone Cooling Can Improve the Growth and Morphology of Spinach in Warm Climates, but the Effectiveness Varies with Cultivar and Air Temperature

Spinach growth is influenced by both air and root-zone temperatures, with high yields observed at specific temperatures depending on the season and cultivar [35]. Controlling the root-zone solution temperature in hydroponics is generally considered easier than managing the air temperature of an entire greenhouse [36]. Optimal growth of spinach plants depends primarily on the surrounding temperature, both air temperature and root-zone temperature, and the specific optimal temperature range varies with species and even cultivars within a species, as evidenced in this study.
In our study, under high greenhouse air temperatures (average 29.3 °C) in Cycle 1, the four spinach cultivars responded distinctly to root-zone cooling. ‘Mandolin’ showed significant increases in shoot and root growth when the root zone was cooled. These results support previous studies on butterhead lettuce [37], where a consistent nutrient solution temperature between 14 °C and 18 °C significantly enhanced its growth and quality, and another study in cucumber [19], which reported that cooler root temperatures (20 °C) led to greater biomass production. However, in ‘SV2157’, all plants grew well, regardless of root-zone cooling, indicating high heat tolerance and the ability to grow under high air and root-zone temperatures. ‘Lakeside’ and ‘Hammerhead’ did not show significant growth increases with root-zone cooling in Cycle 1. Interestingly, in Cycle 2, when the weather was cooler, with an average air temperature of 24.65 °C, ‘Lakeside’ and ‘Hammerhead’ produced higher shoot FW, shoot DW, and root DW, while ‘Mandolin’ and ‘SV2157’ showed no significant increases in growth compared to that in Cycle 1. These results may suggest that ‘Lakeside’ and ‘Hammerhead’ are less heat tolerant than ‘Mandolin’, while ‘SV2157’ is the most heat tolerant among the four cultivars. Additionally, our findings highlighted that ‘Lakeside’ and ‘Hammerhead’ were more sensitive to both air and root-zone temperatures and achieved better growth under low air and root-zone temperatures.
Root-zone cooling likely reduces stress in the root zone and allows normal leaf expansion and broader leaf development. Additionally, cooler root temperatures enhance cytokinin synthesis [38], which promotes cell division and expansion in leaves. The increase in leaf area ultimately contributed to improved photo-assimilation and higher biomass production [6]. Similar effects have been observed in previous studies, where lower root temperatures increased leaf width in Cyclamen persicum [39] and expanded leaf area in Lactuca sativa [40] and cucumber (Cucumis sativus) at root-zone cooling temperatures of 22 °C and 25 °C [19]. In our study, LA increased under root-zone cooling temperatures compared to that in the control only in ‘Mandolin’. However, LA in ‘SV2157’, ‘Lakeside’, and ‘Hammerhead’ were not significantly different among treatments, indicating that RZT does not influence the leaf expansion of these cultivars. In addition, root-zone cooling significantly influenced morphological parameters, as LL and LW increased under RZT24 and RZT21 compared to those in the control, validating the role of leaf expansion under cooler root-zone conditions.

4.2. Root-Zone Cooling Has the Potential to Improve Spinach Physiology, Reduce Stress, and Protect Phytochemical Properties Under Warm Climate

Based on the growth and morphological data, it has been confirmed that the plants in Cycle 2 exhibited consistent and healthy growth, while the plants in Cycle 1 showed significant stress. Consequently, this section focuses on the physiological and stress indicator outcomes observed in Cycle 1 only.
The maximum quantum efficiency of photosystem II, Fv/Fm, is often used to evaluate stress. High temperatures can reduce the Fv/Fm ratio by damaging PSII and the heat-sensitive component of the photosynthetic apparatus, leading to decreased electron transport rates and impaired photochemical efficiency [41]. The ideal Fv/Fm value is approximately 0.83, representing the maximum efficiency of PSII in converting light energy into chemical energy under non-stressed conditions [42]. Our study indicated that Fv/Fm values were equally lower than the optimum regardless of root-zone cooling and cultivar, possibly due to both the high air temperature and high light intensity in the middle of the day. However, there were cultivar-specific differences in Fv/Fm. These differences may indicate that ‘Hammerhead’ was more stressed than ‘Mandolin’ and ‘SV2157’. This cultivar-specific response supports observations in crops like sweet cherry and cucumber, where root-zone cooling enhanced growth without significantly altering Fv/Fm values among treatments [19,43]. This suggests that Fv/Fm may not be sensitive enough to detect the differences caused by root-zone cooling treatments in the spinach cultivars in this study.
Exposure to high temperatures can degrade leaf pigments, particularly chlorophyll, by damaging chloroplasts through mechanisms such as disruption of membrane fluidity, protein denaturation, and increased production of reactive oxygen species (ROS), ultimately reducing photosynthetic activity [44]. Although carotenoids are more heat-resistant than chlorophyll, they can still be affected by high temperatures, potentially altering plant coloration, whereas the effect of high temperatures on anthocyanin content varies among plant species [45,46]. In our study, significant differences in leaf pigment contents were observed among cultivars. ‘SV2157’ consistently exhibited the highest levels of total chlorophyll, anthocyanin, and carotenoids, indicating a genetic influence on pigment accumulation and stress tolerance. Among leaf pigments, only anthocyanin was affected by root-zone temperature, suggesting that anthocyanin is more responsive to heat stress. These findings highlight the influence of root-zone temperature on pigment biosynthesis and its potential implications for improving plant resilience under heat stress.
Although both phenolics and flavonoids are secondary metabolites involving stress responses, TPC showed significant differences across treatments and cultivars, while TFC did not exhibit any significant differences. The highest TPC levels were noted in ‘Hammerhead’ and ‘Lakeside’ under control treatment, supporting the role of phenolics as stress indicators, where TPC increases under high temperatures to enhance plant defense by neutralizing ROS [47]. The absence of differences in TPC among treatments in ‘SV2157’ further suggested its heat tolerance. However, the lack of significant changes in TFC among cultivars and treatments may be attributed to the plants’ metabolic prioritization. Under high temperatures, spinach may allocate more resources toward phenolic production rather than flavonoid production due to energy constraints [45]. Additionally, the enzymes involved in flavonoid biosynthesis might be less responsive to temperature fluctuations, leading to minimal changes in TFCs.
High temperatures can trigger ROS accumulation, leading to oxidative damage [48]. In our findings, H2O2, as a typical ROS, was notably higher with control treatment for ‘Mandolin’ and ‘Hammerhead’, indicating that these plants were under oxidative stress. However, H2O2 levels in both ‘Hammerhead’ and ‘Mandolin’ were significantly reduced as a result of root-zone cooling, particularly under RZT24. This result showed that root-zone cooling could reduce stress levels caused by high air temperatures by modulating ROS levels. In ‘SV2157’, the lower H2O2 levels across all treatments again proved that ‘SV2157’ was the most heat-tolerant cultivar among the four cultivars.
Another key marker of oxidative stress in plants is MDA. Specifically, MDA indicates the extent of lipid peroxidation caused by ROS, where elevated MDA levels reflect significant damage to cell membranes due to excessive ROS activity [49,50]. In the present study, increased MDA and cell leakage were more prominent under control treatment than under the other RZTs and followed a similar trend: ‘Hammerhead’ > ‘Lakeside’ > ‘Mandolin’. In contrast, ‘SV2157’ exhibited consistent levels of MDA and cell leakage regardless of RZT treatment. Chadirin et al. [51] reported that a high RZT increases membrane permeability, highlighting the importance of temperature regulation in maintaining cell integrity. Consequently, RZT cooling enhanced cellular stability in all cultivars except ‘SV2157’.

4.3. Optimal Root-Zone Cooling Temperature with Consideration of Electricity Consumption and Plant Performance

Root-zone cooling is a relatively low-input alternative to cooling the entire greenhouse. However, this method results in additional costs. Our findings confirm that a lower root-zone temperature leads to higher electricity consumption. However, it remains uncertain whether a lower root-zone temperature improves plant performance. Results in our study demonstrated cultivar-dependent responses to root-zone cooling: from no significant changes in ‘SV2157’ to no clear benefit in ‘Lakeside’ and ‘Hammerhead’. In ‘Mandolin’, significant improvements in plant growth and reduction of heat stress were observed in RZT24; however, further decreasing the root-zone temperature from 24 °C to 21 °C did not show further improvement in growth and stress indicators. These results suggest that cooling root-zone to 24 °C was sufficient for ‘Mandolin’ in terms of growth and mitigating heat stress. In ‘Lakeside’ and ‘Hammerhead’, there were some indications of stress reduction with root-zone cooling either to 24 or 21 °C; however, the benefits were not obvious, possibly due to the high air temperature, indicating that these two cultivars were more sensitive to heat stress compared to ‘Mandolin’. Nevertheless, considering plant performance in all four cultivars and electricity consumption, cooling to 21 °C is not necessary.

5. Conclusions

Root-zone cooling in hydroponic systems in warm climates significantly influenced plant growth, physiology, and heat stress alleviation in spinach plants, although the impact varied by cultivar. Among the four cultivars, ‘Mandolin’ benefited the most from root-zone cooling, showing improvements in growth and stress indicators when the root-zone temperature was cooled to 24 °C; however, further cooling from 24 °C to 21 °C did not produce greater benefits. ‘SV2157’ performed equally well at all root-zone temperatures in both production cycles, exhibiting strong heat tolerance, superior physiological traits, including increased chlorophyll, carotenoid, and anthocyanin levels compared to those in other cultivars, and the lowest stress levels among all treatments. The positive impact of root-zone cooling in ‘Lakeside’ and ‘Hammerhead’ was unclear, possibly due to the high air temperature; thus, these cultivars were considered less heat tolerant than ‘Mandolin’ and ‘SV2157’. These findings suggest significant variations in heat tolerance among spinach cultivars. Considering growth and energy costs, cooling the root zone to 24 °C is adequate under the conditions of this study. Future studies should focus on screening more spinach cultivars and exploring strategies to optimize the production of heat-sensitive spinach under different air temperatures, as the effectiveness of root-zone cooling varies with air temperature. These investigations will provide a more comprehensive understanding for enhancing spinach production in warm climates.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su17093925/s1. Table S1: Summary of analysis of variance (ANOVA) on the effects of three root zone temperatures (RZT) and four spinach cultivars (CV) on morphological data: third leaf length (LL), third leaf width (LW), leaf number (LN), stem length (SL), and stem diameter (SD); physiological attributes: maximum quantum yield (Fv/Fm), total chlorophylls, total carotenoids, anthocyanin, total phenolics content (TPC), and total flavonoids content (TFC); and stress indicators: mortality, hydrogen peroxide (H2O2), malondialdehyde (MDA), cell leakage in Cycle 2; NS or * indicate not significant or significant at a level of p ≤ 0.05. Figure S1: Experimental layout and randomization process for both cycles. Each of the blocks consisting of three deep water culture (DWC) hydroponic systems and under each DWC four cultivars also were randomized. Figure S2: Photos of representative individual plants of four spinach cultivars (Lakeside, Hammerhead, Mandolin and SV2157) in response to three different root zone temperatures (Control, 24 °C = RZT24, and 21 °C = RZT21).

Author Contributions

Conceptualization, G.N., M.N.E.A.K. and J.M.; resources, G.N.; investigation, M.N.E.A.K. and G.N.; data curation, M.N.E.A.K.; writing—original draft preparation, M.N.E.A.K.; writing—review and editing, M.N.E.A.K., J.M. and G.N.; supervision, G.N.; project administration, G.N.; funding acquisition, G.N. and J.M. All authors have read and agreed to the published version of the manuscript.

Funding

Specialty crop block grant program (SCBG) from the Texas Department of Agriculture (Grant number: GSC2022145).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Daily average air temperatures inside the greenhouse and daily electricity consumption by the chillers under two different root-zone temperature (RZT) treatments: 24 °C (RZT24) and 21 °C (RZT21) in Cycle 1 (A); time course of nutrient solution temperatures and air temperatures on the 14th day after transplanting (13 August 2024, Cycle 1; 9 September 2024, Cycle 2) (B).
Figure 1. Daily average air temperatures inside the greenhouse and daily electricity consumption by the chillers under two different root-zone temperature (RZT) treatments: 24 °C (RZT24) and 21 °C (RZT21) in Cycle 1 (A); time course of nutrient solution temperatures and air temperatures on the 14th day after transplanting (13 August 2024, Cycle 1; 9 September 2024, Cycle 2) (B).
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Figure 2. Shoot fresh weight (A,B), shoot dry weight (C,D), root dry weight (E,F), and total leaf area (G,H) of four spinach cultivars (‘Lakeside’: LS, ‘Hammerhead’: HH, ‘Mandolin’: MD, and ‘SV2157’: SV) grown in hydroponic systems with or without root-zone cooling. The left-side graphs correspond to Cycle 1, and the right-side graphs correspond to Cycle 2. Vertical error bars represent the standard errors (n = 3). Different letters indicate significant differences across the root-zone temperatures in a single cultivar at p ≤ 0.05. NS denotes no significant differences.
Figure 2. Shoot fresh weight (A,B), shoot dry weight (C,D), root dry weight (E,F), and total leaf area (G,H) of four spinach cultivars (‘Lakeside’: LS, ‘Hammerhead’: HH, ‘Mandolin’: MD, and ‘SV2157’: SV) grown in hydroponic systems with or without root-zone cooling. The left-side graphs correspond to Cycle 1, and the right-side graphs correspond to Cycle 2. Vertical error bars represent the standard errors (n = 3). Different letters indicate significant differences across the root-zone temperatures in a single cultivar at p ≤ 0.05. NS denotes no significant differences.
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Figure 3. The maximum quantum yield (Fv/Fm) (A), total chlorophyll (B), anthocyanin (C), total carotenoids (D), total phenolic content (E), and total flavonoid content (F) of four spinach cultivars (‘Lakeside’: LS, ‘Hammerhead’; HH, ‘Mandolin’: MD, and ‘SV2157’: SV) grown under three root-zone temperature treatments (Control, RZT24 ≈ 24 °C, and RZT21 ≈ 21 °C) in Cycle 1. Data were pooled across cultivars or treatments except for total phenolic content (interactive effects). Vertical error bars represent the standard errors (n = 3). Different letters indicate significant differences at p ≤ 0.05, and NS denotes no significant differences.
Figure 3. The maximum quantum yield (Fv/Fm) (A), total chlorophyll (B), anthocyanin (C), total carotenoids (D), total phenolic content (E), and total flavonoid content (F) of four spinach cultivars (‘Lakeside’: LS, ‘Hammerhead’; HH, ‘Mandolin’: MD, and ‘SV2157’: SV) grown under three root-zone temperature treatments (Control, RZT24 ≈ 24 °C, and RZT21 ≈ 21 °C) in Cycle 1. Data were pooled across cultivars or treatments except for total phenolic content (interactive effects). Vertical error bars represent the standard errors (n = 3). Different letters indicate significant differences at p ≤ 0.05, and NS denotes no significant differences.
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Figure 4. Mortality (%) (A), hydrogen peroxide (H2O2) level (B), malondialdehyde (C), and % cell leakage (D) under interactive effects between three root-zone temperatures (Control, RZT24 ≈ 24 °C, and RZT21 ≈ 21 °C) and four spinach cultivars (‘Lakeside’: LS, ‘Hammerhead’: HH, ‘Mandolin’: MD, and ‘SV2157’: SV) in Cycle 1. Vertical error bars represent the standard errors (n = 3). Different letters indicate significant differences across the root-zone temperatures in a single cultivar at p ≤ 0.05. NS denotes no significant differences.
Figure 4. Mortality (%) (A), hydrogen peroxide (H2O2) level (B), malondialdehyde (C), and % cell leakage (D) under interactive effects between three root-zone temperatures (Control, RZT24 ≈ 24 °C, and RZT21 ≈ 21 °C) and four spinach cultivars (‘Lakeside’: LS, ‘Hammerhead’: HH, ‘Mandolin’: MD, and ‘SV2157’: SV) in Cycle 1. Vertical error bars represent the standard errors (n = 3). Different letters indicate significant differences across the root-zone temperatures in a single cultivar at p ≤ 0.05. NS denotes no significant differences.
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Table 1. Average root-zone and air temperatures, dissolved oxygen concentration, daily light integral, electrical conductivity (EC), and pH of the nutrient solution and daily average electricity consumption throughout the two growth cycles.
Table 1. Average root-zone and air temperatures, dissolved oxygen concentration, daily light integral, electrical conductivity (EC), and pH of the nutrient solution and daily average electricity consumption throughout the two growth cycles.
CycleTreatmentTime Frame
(Transplanting to Harveting)		Average Root-Zone Temperature (°C)Average
Air Temperature
(°C)		Dissolved Oxygen
(mg/L)		Daily Light Integral (mol/m2/d)Range
of EC
(mS/cm)		Range of pHAverage Electricity Consumption
(kWh/day)
1Control30 July 2024 to 20 August 202428.0 6.2813.740.99–1.485.86–6.33-
RZT2423.929.37.220.96–1.426.07–6.681.23
RZT2121.1 7.290.89–1.396.10–6.572.67
2Control27 August 2024 to 18 September 202423.9 8.029.861.02–1.446.01–6.54-
RZT2423.724.78.181.00–1.436.03–6.490.83
RZT2120.9 8.270.97–1.386.07–6.661.84
Table 2. Summary of analysis of variance (ANOVA) for Cycle 1 of the effects of three root-zone temperatures (RZT) and four spinach cultivars (CV) on growth parameters: shoot fresh weight (FW), shoot dry weight (DW), root DW, and total leaf area; physiological attributes: maximum quantum yield (Fv/Fm), total chlorophyll, total carotenoids, anthocyanin, total phenolics content (TPC), and total flavonoids content (TFC); and stress indicators: mortality, hydrogen peroxide (H2O2), malondialdehyde (MDA), and cell leakage.
Table 2. Summary of analysis of variance (ANOVA) for Cycle 1 of the effects of three root-zone temperatures (RZT) and four spinach cultivars (CV) on growth parameters: shoot fresh weight (FW), shoot dry weight (DW), root DW, and total leaf area; physiological attributes: maximum quantum yield (Fv/Fm), total chlorophyll, total carotenoids, anthocyanin, total phenolics content (TPC), and total flavonoids content (TFC); and stress indicators: mortality, hydrogen peroxide (H2O2), malondialdehyde (MDA), and cell leakage.
Growth DataPhysiological AttributesStress Indicators
FactorShoot FWShoot DWRoot DWLeaf AreaFv/FmTotal ChlorophyllTotal CarotenoidsAnthocyaninTPCTFCMortalityH2O2MDACell Leakage
(g)(g)(g)(sq. cm) (μg/mL)(μg/mL)(mg/g FW)(GAE/g FW)(CE/g FW)(%)(μmol/g FW)(nmol/g FW)(%)
RZT**********NSNSNS****NS************
CV*************************NS***********
RZT × CV*******NSNSNSNS***NS************
Note: NS indicates not significant, and *, **, and *** indicate significance at p levels of ≤0.05, 0.01, and 0.001, respectively.
Table 3. Main effects and analysis of variance (ANOVA) summary of three root-zone temperatures (RZTs) and four spinach cultivars (CVs) on morphological parameters: Third leaf length (LL), third leaf width (LW), leaf number (LN), stem length (SL), and stem diameter (SD) in Cycle 1.
Table 3. Main effects and analysis of variance (ANOVA) summary of three root-zone temperatures (RZTs) and four spinach cultivars (CVs) on morphological parameters: Third leaf length (LL), third leaf width (LW), leaf number (LN), stem length (SL), and stem diameter (SD) in Cycle 1.
FactorLL
(cm)		LW
(cm)		LNSL
(cm)		SD
(mm)
Root-zone temperature (RZT)
Control12.52 ± 1.9 b z6.62 ± 1.2 b17.45 ± 3.62.23 ± 0.44.61 ± 0.9 b
RZT2414.79 ± 2.4 a8.16 ± 1.1 a18.17 ± 3.42.31 ± 0.75.15 ± 0.9 a
RZT2112.52 ± 2.4 ab8.16 ± 1.5 a18.26 ± 3.02.36 ± 0.55.14 ± 0.8 ab
Cultivar (CV)
Lakeside11.45 ± 1.3 b6.65 ± 1.1 c16.22 ± 0.9 b1.92 ± 0.34.61 ± 0.6 b
Hammerhead14.11 ± 2.1 a7.21 ± 0.9 bc15.69 ± 2.2 b2.52 ± 0.64.03 ± 0.4 b
Mandolin15.17 ± 2.7 a8.12 ± 1.9 ab19.85 ± 3.7 a2.55 ± 0.65.48 ± 0.5 a
SV215714.60 ± 1.5 a8.61 ± 1.0 a20.06 ± 3.1 a2.22 ± 0.55.75 ± 0.6 a
ANOVA summary
RZT***NSNS*
CV********NS**
RZT × CVNSNSNSNSNS
Note: NS indicates not significant, and *, **, and *** indicate significance at p levels of ≤0.05, 0.01, and 0.001, respectively. z The values are the means ± standard deviations; different letters indicate significant differences, and values without any letters showed no significant differences.
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MDPI and ACS Style

Khan, M.N.E.A.; Masabni, J.; Niu, G. Cultivar-Specific Responses of Spinach to Root-Zone Cooling in Hydroponic Systems in a Greenhouse Under Warm Climates. Sustainability 2025, 17, 3925. https://doi.org/10.3390/su17093925

AMA Style

Khan MNEA, Masabni J, Niu G. Cultivar-Specific Responses of Spinach to Root-Zone Cooling in Hydroponic Systems in a Greenhouse Under Warm Climates. Sustainability. 2025; 17(9):3925. https://doi.org/10.3390/su17093925

Chicago/Turabian Style

Khan, Md Noor E Azam, Joseph Masabni, and Genhua Niu. 2025. "Cultivar-Specific Responses of Spinach to Root-Zone Cooling in Hydroponic Systems in a Greenhouse Under Warm Climates" Sustainability 17, no. 9: 3925. https://doi.org/10.3390/su17093925

APA Style

Khan, M. N. E. A., Masabni, J., & Niu, G. (2025). Cultivar-Specific Responses of Spinach to Root-Zone Cooling in Hydroponic Systems in a Greenhouse Under Warm Climates. Sustainability, 17(9), 3925. https://doi.org/10.3390/su17093925

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