2013 Volume 82 Issue 4 Pages 322-327
Low-cost technology is needed to alleviate high-temperature injury for high-yield greenhouse tomato production. To acquire information about the physiological and morphological effects of root-zone cooling, we grew young tomato plants for 2 weeks in nutrient solution held at about 25°C, considered to be the optimum temperature for tomato plants. We investigated plant growth, nutrient uptake, root activity (xylem exudation and root respiration rate), root indole-3-acetic acid (IAA) concentration, and internal root structure. The root-zone temperature was maintained at 24.7°C by cooling, while the air temperature and control temperatures were higher than optimum (30.8 and 33.7°C, respectively). Root-zone cooling increased the relative growth rate (RGR) of roots compared with the control, followed by shoot RGR. Root IAA was positively correlated with root RGR. Root-zone cooling increased Ca and Mg uptake as well as root xylem exudation and respiration. It also advanced the development of the internal structure of the xylem near the root tip. Thus, possibly by increasing root activity and root IAA, root-zone cooling promoted root growth and nutrient uptake mediated by the development of the root xylem, and thus shoot growth. These results suggest a physiological and morphological mechanism of growth enhancement by root-zone cooling under high air temperature conditions.
In tomato cultivation in the greenhouse, avoidance of high temperatures is important in summer in Japan because summer temperatures exceed the optimum for tomato production. High temperatures reduce yields by reducing pollen viability and fruit set (Sasaki et al., 2005; Sato et al., 2006), causing cuticle cracking (Dorais et al., 2004) and decreasing root activity (Gosselin and Trudel, 1983a, b; Klock et al., 1997).
Cooling technologies have been developed to permit year-round production to alleviate high temperature injury to tomato. Heat exchanger (De Gelder et al., 2005), pad-and-fan, and fog (Peet and Welles, 2005) cooling systems are sometimes used, but they are expensive to run or not particularly effective under high humidity. Therefore, effective and economic cooling systems are needed. These are also important for decreasing CO2 emission to prevent global warming.
One such system, the root-zone cooling technique, is proposed. In the case of tomato, when the root-zone temperature was cooled to 25°C under high air temperatures, vegetative growth was improved by increasing leaf area and plant height compared with uncooled plants, and fruit yield was also increased (Fujishige et al., 1991; Nkansah and Ito, 1994). In addition Sasaki and Itagi (1989) reported that fruit yield was increased by root-zone cooling at 20°C in summer tomato production. These increases of growth and fruit yield were explained by enhancing photosynthetic activities (Nkansah and Ito, 1995a), improving water and nutrient uptake (Klock et al., 1997; Nkansah and Ito, 1995b), and increasing root respiration rate (Klock et al., 1997). These parameters were maximized at around 25°C root-zone temperature. However, little knowledge comparing the partial difference in growth and the nutrient content by root-zone cooling is available, and physiological or morphological information is also insufficient.
It is important to obtain fundamental knowledge about the effects of root-zone cooling on plant growth as well as to develop a practical root-zone cooling system. Therefore, we investigated physiological and morphological changes at the early developmental stage of tomato plants.
Seeds of tomato (Solanum lycopersicum L.) ‘Momotaro-Yoku’ (Takii seed Co., Ltd., Kyoto, Japan) were sown in 72-cell trays filled with vermiculite, and seedlings were grown for 3 weeks in a growth chamber with day/night air temperatures of 25/20°C. The seedlings were then transplanted into a nutrient-film-technique hydroponic system in a glasshouse on 6 July 2012. The glasshouse was ventilated when the temperature exceeded 25°C. The nutrient solution, renewed every 2 weeks, contained 82 (μg·g−1) N, 15 P, 134 K, 55 Ca, 12 Mg, 1.3 Fe, 0.4 Mn, 0.3 B, 0.04 Zn, 0.01 Mo, and 0.01 Cu. The root-zone cooling treatment was started 5 days after transplanting, with the solution temperature maintained at around 25°C, and continued for 14 days. The root zone of half of the seedlings remained uncooled as a control. The average air temperature in the glasshouse during the treatment period was 30.8°C, and the maximum and minimum temperatures were 38.3°C and 24.6°C, respectively. The nutrient solution temperature averaged 24.7°C in the cooling treatment and 33.7°C in the control.
Measurement of xylem exudation, root respiration, and dry weightPlants were assessed on days 0 (with 7–9 leaves), 7 (10–12 leaves), and 14 (13–16 leaves) of cooling treatment. Plants (n = 6) were cut off below the first true leaves, the cotyledons were removed, and we collected the xylem exudate for 10 min in absorbent cotton for later weighing (Yamaguchi et al., 1995). Some well-developed lateral roots (0.1 to 0.6 g of total dry weight) from base to tip (n = 6 plants) were immersed in 500 mL stirred nutrient solution, which was the same solution as for cultivation, and the rate of oxygen consumption was measured with an oxygen electrode (D-55; HORIBA, Ltd., Kyoto, Japan) at 30°C to calculate the root respiration rate. Xylem exudation and the root respiration rate were measured between 9:00 and 15:00. Shoots and roots (n = 8) were dried at 80°C for at least 48 h and then weighed for calculation of the relative growth rate (RGR), which was calculated from the equation:
Fifty milligrams of dried samples of shoots and roots were pulverized and ashed in 10 mL nitric acid and 2 mL hydrogen peroxide at 180°C for 20 min in a microwave ashing system (START D; Milestone-general Co., Ltd., Kanagawa, Japan). P, K, Ca, and Mg were measured by inductively coupled plasma atomic emission spectrometry (SPS7700; Hitachi High-Tech Science Corporation Co., Ltd., Tokyo, Japan). N was measured using a CN analyzer (JM1000CN; J-Science Lab Co., Ltd., Kyoto, Japan).
Quantification of IAAFor analysis of indole-3-acetic acid (IAA), about 1 g fresh roots was homogenized in liquid nitrogen and placed in 10 mL methanol/water/formic acid (15 : 4 : 1, v/v/v). 13C6-labeled IAA (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) was added to the extracts as an internal standard. After overnight extraction at 4°C, solids were separated by centrifugation and re-extracted for 30 min in 10 mL of the same extraction solution. To remove interfering compounds, extracts were first passed through an Oasis HLB column (200 mg; Waters, Milford, MA, USA) equilibrated with 1 M formic acid. After further washing with 5 mL extraction solvent, the combined eluate was evaporated; the residue was dissolved in 5 mL of 1 M formic acid, and the solution applied to an Oasis MCX column (150 mg; Waters) equilibrated with 1 M formic acid. After further washing with 5 mL of 1 M formic acid, the IAA was then eluted with 5 mL methanol. The eluate was evaporated, and the residue was dissolved in 3 mL of 1% acetic acid. The solution was applied to an Oasis WAX column (60 mg; Waters) equilibrated with 1% acetic acid. After further washing with 3 mL of 1% acetic acid and then with 2 mL methanol, IAA was eluted with 6 mL of 80% methanol containing 1% acetic acid. The eluate was evaporated at 40°C under vacuum. The residues were then dissolved in water/methanol/acetic acid (80 : 19.95 : 0.05, v/v/v) and analyzed by high-performance liquid chromatography (HPLC)/tandem quadrupole mass spectrometry (MS/MS). The HPLC-MS/MS system consisted of a Prominence 20A Series HPLC (Shimadzu Co., Ltd., Kyoto, Japan), a 3200 QTrap LC/MS/MS System (AB Sciex, Framingham, MA, USA), and an electrospray interface. The purified samples were injected onto a Shim-pack XR-ODS column (2.2 μm, 100 mm × 2.0 mm; Shimadzu) at 45°C and eluted at a flow rate of 0.2 mL·min−1. For chromatographic separation, mobile phase A was water/methanol/acetic acid (80: 19.95: 0.05, v/v/v) and mobile phase B was methanol. The initial conditions were 100% A for 2 min; linear change to 40% A/60% B over 7 min; to 100% B over 10 min; and held at 100% B for 3 min. Before each analytical run, the column was equilibrated with mobile phase A for 10 min. IAA was quantified by multiple reaction monitoring of the protonated intact precursor ion [M + H]+ and a specific product ion, using the following mass transitions: [13C6]IAA, 182.1 >136.2; IAA, 176.1 > 130.1. Data were analyzed using Analyst v. 1.4.2 software (AB Sciex). Concentrations were calculated from the peak area of the endogenous compound, relative to the area of the internal standard. Three biological replicates were analyzed for all samples.
Root morphologyOn day 14, four lateral root samples near the root tip from each treatment were fixed in FAA (40% formaldehyde/acetic acid/ethanol/water = 1 : 1 : 9 : 9). Then samples were dehydrated in a graded ethanol series (70%, 80%, 90%, 100%), and embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany). The embedded samples were sectioned on a microtome at 2 μm and stained with toluidine blue. The cross sections were then observed and photographed under an optical microscope (CX41; Olympus Co., Ltd., Tokyo, Japan).
Although the shoot dry weight in the root-zone cooling treatment was significantly less than that in the control on day 7, the difference had disappeared by day 14 (Fig. 1A). There was no difference in shoot RGR during day 0 to 7, but RGR was significantly greater in the cooling treatment during day 7 to 14 (Fig. 1B). Root dry weights did not show significant differences between treatments on days 7 or 14 (Fig. 1C). Root RGR was significantly greater in the cooling treatment during day 0 to 7, but there was no difference during day 7 to 14 (Fig. 1D).
Effects of root-zone cooling treatment and time on (A) shoot dry weight (DW), (B) shoot relative growth rate (RGR), (C) root DW, and (D) root RGR in tomato plants. Data are the means (±SE) of eight independent measurements. *: significant at P < 0.05 by t-test.
The rate of xylem exudation did not change over time in the control, but it was doubled on day 7 and quadrupled on day 14 in the cooling treatment (Fig. 2A). The root respiration rate increased in both the control and treatment groups on day 7 and then decreased in both on day 14. That on day 14 was significantly higher in the cooling treatment group (Fig. 2B).
Effects of root-zone cooling treatment and time on (A) xylem exudation rate and (B) root respiration rate in tomato plants. Data are the means (±SE) of six independent measurements. *: significant at P < 0.05 by t-test.
IAA concentration was determined in roots under root-zone cooling treatment in order to show the relationship between IAA concentration and root growth. Although there was no significant change in root IAA concentration over time in the control, the concentration increased significantly on day 7 and decreased on day 14 in the cooling treatment group. There was a significant positive correlation between IAA concentration and root RGR (Fig. 3).
Correlation between root relative growth rate (RGR) and root IAA concentration in control and root-zone cooled tomato plants after 7 and 14 days of treatment. Data are means (±SE) of three (root IAA concentration) or eight (root RGR) independent measurements. *: significant correlation at P < 0.05.
Effect of root-zone cooling on mineral concentration was investigated in shoots and roots (Table 1). Shoot Ca and Mg concentrations were significantly lower on days 7 and 14 than on day 0 in the control. In contrast, these decreases were not observed with cooling treatment, and thus were higher than in the control. Shoot N concentration was also significantly lower on days 7 and 14 than on day 0 in the control but not in the cooling treatment group. Shoot P and K concentrations were significantly lower on days 7 and 14 than on day 0 in both treatments. Root Ca and Mg concentrations showed the opposite tendency: they were significantly higher on days 7 and 14 than on day 0 in the control but unchanged with the cooling treatment, and thus lower than in the control. Root K concentration was significantly lower only on day 7 in the control and was unchanged in the cooling treatment group. Root P concentration was significantly lower only on day 14 with cooling treatment. Root N concentration was unchanged. The total uptake of N, Ca, and Mg, which were their content per plant by multiplying each concentration by dry weight, was significantly greater in the cooling treatment than in the control group on day 14.
| Days | Shoot conc. (mg·g−1 DW) | Root conc. (mg·g−1 DW) | Uptake (mg per plant) | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N | P | K | Ca | Mg | N | P | K | Ca | Mg | N | P | K | Ca | Mg | ||
| 0 | Control | 39.4 az | 5.9 a | 51.2 a | 16.5 a | 5.6 a | 41.8 a | 8.7 a | 32.2 a | 5.2 b | 7.4 b | 143.2 a | 22.0 a | 178.5 a | 56.4 a | 20.8 a |
| 7 | Control | 35.1 b | 4.7 b | 41.4 b | 15.9 b | 4.6 b | 37.3 a | 8.3 ab | 23.4 b | 12.0 a | 9.7 a | 354.9 b | 48.9 b | 406.1 b | 157.3 b | 49.5 b |
| Cooling | 38.0 ab | 5.0 b | 40.4 b | 18.6 a | 5.6 a | 43.4 a | 7.2 bc | 33.0 a | 4.5 b | 7.2 b | 328.1 b | 44.5 b | 338.6 b | 149.3 b | 48.8 b | |
| 14 | Control | 35.0 b | 4.8 b | 40.9 b | 16.0 b | 4.5 b | 38.8 a | 8.2 ab | 32.4 a | 13.0 a | 9.2 a | 672.9 c | 95.8 c | 766.2 c | 300.8 c | 90.9 c |
| Cooling | 37.9 ab | 5.4 ab | 38.3 b | 21.5 a | 5.4 a | 41.0 a | 6.2 c | 35.0 a | 5.3 b | 6.5 b | 807.2 d | 112.7 c | 786.4 c | 426.8 d | 112.4 d | |
We investigated not only dry weight or root activity, but also internal root structures by observation of root sections. Histological examinations revealed that xylem vessel cells near the root tip were clearly larger in the cooling treatment group (Fig. 4). That change was observed among all sections and typical images are shown in the figure.
Cross-sections of root near the root tip of tomato plants from (A) the control and (B) root-zone cooling treatment on day 14. Scale bar = 50 μm. Arrows indicate xylem vessels.
Photosynthetic activities are decreased at day/night air temperatures of 40/23°C in young tomato plants (Nkansah and Ito, 1995a) and RGR is also decreased over 30°C air temperature (Gent, 1986). Pollen viability and release of pollen grains were reduced at 32/26°C (Sato et al., 2006). Tomato fruit set was reduced at 34/20°C (Sasaki et al., 2005). Therefore, at an average of 30.8°C, the air temperature during the treatment period in our study was high enough to injure tomato plants.
The rates of photosynthesis and growth were decreased at a root-zone temperature of 30°C under high air temperature conditions (Nkansah and Ito, 1995a). In other plants, they were also decreased at high root-zone temperatures (Benoit and Ceustermans, 2001; Du and Tachibana, 1994; He et al., 2001; Wang and Tachibana, 1996). Although there have been studied at optimum air temperature, root-zone temperature exceeding 30°C reduced nutrient uptake (Tindall et al., 1990) and yield (Gosselin and Trudel, 1983a). In our experiment, the average temperature was 33.7°C in the control solution, which is supposed to cause high temperature injuries, but was 24.7°C in the cooling treatment, close to the optimum (Klock et al., 1997; Nkansah and Ito, 1995a, b).
Cooling treatment increased xylem exudation and the root respiration rate (Fig. 2), which are indicators of root activity (Yamaguchi et al., 1995). These results indicate improved transport of water and nutrients (Klock et al., 1997; Yamaguchi et al., 1995) and support the report that water uptake by roots was maximized at 25°C (Nkansah and Ito, 1995b).
Root IAA was high on day 7 of cooling treatment. This result suggests a response of roots to the optimal root-zone temperature. There was a positive correlation between root IAA and RGR (Fig. 3). In Arabidopsis, root IAA promotes root growth (Novickienė et al., 2010; Ohashi-Ito et al., 2013). Therefore, this result confirms the promotion of root growth during day 0 to 7 of cooling treatment and agrees with the previous study (Nkansah and Ito, 1994). On the other hand, root IAA was decreased on day 14. Pressman et al. (1997) showed that root RGR was decreased with root growth and our result agrees with this observation. Cooling treatment promoted the development of xylem tissue near the root tip (Fig. 4). Although we could find no information on the effect of root-zone temperature on root morphology, the promotion of xylem development by auxin previously reported in Arabidopsis (Deng et al., 2012; Ohashi-Ito et al., 2013) could explain that the transient increase in IAA promoted root xylem development.
Cooling treatment promoted shoot Ca and Mg concentrations and total uptake of Ca, Mg, and N (Table 1). These results are supported by a previous study (Nkansah and Ito, 1995b). Although there have been studied at the optimum air temperature, similar tendencies were reported (Kabu and Toop, 1970; Tindall et al., 1990). On the other hand, root Ca and Mg concentrations were reduced. Although we could find no information on the effect of root-zone cooling on mineral concentrations in roots, these opposite results in shoots and roots suggest that the transport of Ca and Mg within the plant might be promoted by increased root activity and xylem development at the optimal root-zone temperature.
Shoot dry weight on day 7 of cooling treatment was lower than the control (Fig. 1A). Although we could find no information about shoot growth suppression by root-zone cooling, the change of root-zone temperature in a short time might cause shoot growth to be suppressed transiently. Shoot RGR was higher during day 7 to 14 of cooling treatment (Fig. 1B). This result suggests that shoot growth was promoted as a consequence of enhanced root activities and nutrient uptake, and agrees with a previous study (Nkansah and Ito, 1994). In this research, cooling treatment was continued for 14 days. Therefore, further study is needed to reveal the effect of cooling for more than 14 days.
Our results show that root-zone cooling increased root activity and transiently enhanced root IAA, which in turn promoted root growth and nutrient uptake mediated by the development of the root xylem, and thus promoted shoot growth. This study could add physiological and morphological knowledge about the mechanism of growth promotion by root cooling at high air temperature, and provide scientific evidence for the effectiveness of cooling.
