INTRODUCTION
Research rationale
Climate change has increasingly impacted the human environment, nature, and wildlife, with agricultural plants being particularly vulnerable These crops are crucial for the economies of many countries and play a significant role in global trade, yet they are highly sensitive to weather fluctuations Variations in rainfall, extreme temperatures, and severe weather events can drastically affect local crop yields Moreover, unexpected climate changes pose a serious threat to agricultural production As plants are sessile organisms, they constantly face shifts in temperature and other abiotic factors Low temperatures, particularly chilling stress (1–10°C), significantly disrupt cellular physiology, leading to oxidative stress and an imbalance in reactive oxygen species, ultimately resulting in cell and plant death (Lukatkin and Anjum 2014).
Tropical and subtropical plants like cucumber (Cucumis sativus L.) are highly sensitive to frost, thriving best at temperatures above 20°C Low temperatures severely hinder their seedling establishment, physiology, and overall production The severity of chilling injury varies based on air temperature, duration of exposure, and the plant's growth stage, often becoming evident once plants are moved to optimal conditions Chilling temperatures lead to the degradation of membrane lipids in sensitive plants, compromising their structural integrity.
Low-temperature stress alters the composition of photosynthetic pigments in cell membranes, resulting in decreased leaf stomatal conductance and reduced photosynthesis This reduction in CO2 fixation rates leads to an excessive accumulation of reactive oxygen species and heightened activity of antioxidant enzymes.
Low-temperature stresses significantly limit horticultural production in Taiwanese greenhouses during winter, particularly affecting chilling-sensitive plants These chilling temperatures lead to slowed growth, especially in susceptible species, resulting in delayed development and an extended growing season For cucumbers, low temperatures cause delays in growth, a reduction in the formation of new plant organs, altered root structures, and decreased rates of flowering, fruiting, and seed filling This study highlights the detrimental effects of chilling injuries on cucumber seedlings, demonstrating that low temperatures disrupt essential physiological processes, including water regulation, mineral nutrition, photosynthesis, respiration, and metabolism.
Research’s objectives
This study aims to examine the impact of low temperatures on the growth of cucumber seedlings, providing valuable insights for farmers to select the most suitable cucumber cultivars for winter cultivation.
Research questions and hypotheses
• This study aims to solve the following questions:
1 What are the methods to identify the impact of low temperature on cucumber seedlings?
2 How do cucumber seedlings change after chilling treatment?
3 Which cucumber cultivars is the coldest resistance?
1 The cucumber seedlings are affected by chilling treatment
2 There are differences between six cucumber cultivars on the cold resistance
1 The cucumber seedlings are not affected by chilling treatment
2 There are no differences between six cucumber cultivars on the cold resistance.
Limitations
This study faced limitations, including a short internship period and a lengthy duration for each experiment focused on plant growth Additionally, the results varied due to the differing growth rates and environmental adaptations of various cucumber cultivars.
Definitions
Chilling temperatures can severely impact plants in temperate climates, often resulting in reduced yields or complete crop failure due to direct damage or delayed maturation Cucumber cultivars are particularly vulnerable, experiencing chilling injury, which affects chilling-sensitive species that struggle to withstand low temperatures Cold resistance refers to the ability of plants in their vegetative state to endure chilling conditions without compromising their future growth and development (Lukatkin et al 2012).
LITERATURE REVIEW
An overview about six cucumber cultivars
The following Table 2.2 is 6 cucumber cultivars information (Cucumis sativus L.) In this project, the cucumbers were cultivated from seed to the seedlings which have two true leaves as for test
Table 2.2 Six cucumber cultivars information
Cultivar Rate of female flower (%)
High yield, low temperature resistance
Cuigu Multi female Taiwan 24 More female, strong branching Kappa summer no.7 30-50 Japan 21-22 heat-resistant
Nearly all females Japan 20-22 good fruiting rate
Approximately 100% female rate and good fruit rate in the first stalk
Effect of chilling on the physiological processes in chilling-sensitive plants
Low-temperature incubation of chilling-sensitive plants disrupts various physiological processes, including water regulation, mineral nutrition, photosynthesis, respiration, and overall metabolism (Lukatkin et al 2012) This research primarily aims to investigate the effects of low temperatures on photosynthesis.
Photosynthesis is highly sensitive to cold stress, leading to reduced growth and decreased plant productivity at low temperatures This complex process involves various components, including CO2 reduction and photosynthetic photosystems.
Photosystem II is identified as the most cold-sensitive component of the electron transport system Research shows that chilling stress leads to a significant reduction in the maximum quantum yield of PSII (Fv/Fm), indicating damage to photochemical reactions and the inhibition of electron transfer.
Photosystems are essential protein complexes that play a critical role in photosynthesis by facilitating the absorption of light and the transfer of energy and electrons These units are located in the thylakoid membranes of chloroplasts in plants and in the cytoplasmic membrane of photosynthetic bacteria.
Photosystem II (PSII) is the initial protein complex in the light-dependent reactions of oxygenic photosynthesis, situated in the thylakoid membrane of plants It plays a crucial role by using enzymes to capture photons of light, energizing electrons that are then transferred through various coenzymes and cofactors to convert plastoquinone into plastoquinol This process involves oxidizing water to produce hydrogen ions and molecular oxygen, while PSII replenishes lost electrons through the splitting of water, providing the necessary electrons for the overall photosynthesis process.
Chilling-sensitive plants experience a decrease in photosynthesis rates during and after chilling periods, primarily due to lower temperatures and extended chilling durations This reduction in photosynthetic activity can persist even after the plants are moved to warmer conditions (Kingston et al 1999) The physiological factors contributing to this suppression include inhibited phloem transport of carbohydrates from the leaves, limitations in stomatal function, and damage to the photosynthetic apparatus.
The water-splitting complex of photosystem II produces superoxide (O2 -), a reactive oxygen species (ROS) that disrupts electron transport and energy storage, affecting the synthesis of key enzymes in the Calvin and C4 cycles Cold-sensitive plant species exhibit a narrower temperature range for leaf photosynthesis compared to cold-tolerant species (Yamori et al 2009) Chilling sensitive plants in light significantly impacts their photosynthetic apparatus more than chilling in darkness (Szalai et al 2006) Photoinhibition, the reduction of photosynthetic activity due to excessive light during chilling, intensifies with lower temperatures and higher light intensity (Greer, 1995) Although photosystem II is the primary site of photoinhibition, it has been found that damage can also occur at relatively low light and temperature levels, particularly affecting photosystem I (Sonoike).
Chilling temperatures can reduce photosynthesis due to photo-oxidative damage to chloroplast membranes, leading to increased lipid peroxidation and degradation of essential pigments like chlorophyll, carotene, and xanthophylls This damage is caused by activated oxygen species and is linked to decreased antioxidant activity in plant tissues.
Cell membrane changes
Low temperatures significantly alter the physical properties of plant cell membranes, particularly in sensitive species Chilling causes a reduction in membrane elasticity and lipid composition, resulting in decreased lipid fluidity This decline in fluidity negatively impacts the activity of key membrane-bound enzymes, such as H+-ATPase, while simultaneously increasing the lateral diffusion of phospholipids.
Chilling injury, caused by low temperatures, directly impacts cellular components, leading to alterations in membrane characteristics and enzyme structures (Quinn, 1988; Kasamo et al., 1992; Koster et al., 1994; Kasamo et al., 2000) These effects can be reversed if plants are moved to warmer conditions shortly after exposure, before visible damage occurs Membrane deterioration is considered a key factor in the physiological and visible signs of chilling injury Prolonged chilling results in compromised membrane integrity, increased electrolyte leakage, reduced mitochondrial oxidative activity, elevated activation energy for membrane-bound enzymes, decreased photosynthesis rates, metabolic disruptions, and toxic substance accumulation, ultimately manifesting the symptoms of chilling injury (Lyons, 1973).
Membranes are dynamic structures that support numerous biochemical and biophysical reactions They are also major targets of environmental stresses (Leshem,
Chilling impairments lead to significant alterations in metabolic processes, including decreased enzymatic activities, reduced photosynthetic capacity, and changes in membrane fluidity These changes often result in increased membrane permeability, compromising membrane integrity and cell compartmentation under chilling conditions Elevated rates of solute and electrolyte leakage in chilled tissues serve as indicators of membrane damage, with leakage points arising from the formation of membrane domains with varying configurations due to cold exposure.
11 induced changes in lipid phases (Leshem, 1992), or from damage of membrane, particularly as regards lipids (Harwood, 2005)
Malondialdehyde, a key indicator of oxidative damage from stress-induced lipid peroxidation, is commonly used to assess cellular oxidative harm under environmental stress Low temperatures disrupt the metabolism of reactive oxygen species (ROS), leading to their accumulation and the degradation of essential scavenging enzymes like SOD, CAT, POD, and APX Various stress factors are known to elevate ROS production in cells, with photosynthetic electron transport being a primary source The generation of ROS is inevitable during aerobic photosynthesis, and environmental stress significantly intensifies this production Consequently, the dark reactions of photosynthesis are downregulated, resulting in an over-reduction of the electron transport chain and an increase in harmful ROS such as superoxide (O2 -) and hydrogen peroxide (H2O2 -).
The accumulation of reactive oxygen species (ROS), including superoxide (O2 -) and hydrogen peroxide (H2O2 -), during chilling conditions is linked to chloroplast damage While a low level of ROS is necessary for plant functions, excessive levels can result in harmful effects such as lipid peroxidation and the oxidation of proteins and DNA This lipid peroxidation caused by high ROS levels can lead to structural abnormalities and dysfunction in cells.
METHODS
List of instruments
All equipment and machines used in this study are listed in Table 3.1 below
Table 3.1 Name and commercial company of all instruments used in this study
Model LBG-1000 Lead-Biotech Instruments Co., Ltd.,
Taichung, Taiwan Chlorophyll Fluorometer, Mini-Pam, Walz,
Mettler Toledo™, EL20 Benchtop pH Meter Switzerland
Thermolyne/Maxi Mix II USA
Orbital shaker os701 Kansin instruments Co., Taiwan
High-Speed Refrigerated Centrifuge, CR
List of chemicals
All chemicals used in this study are shown below
Table 3.2 Properties of liquid nitrogen
Appearance Colorless gas or liquid
Table 3.3 Properties of trichloroacetic acid (TCA)
Appearance Colorless to white, crystalline solid
Table 3.4 Properties of thiobarbituric acid
Materials
All cucumber cultivars used in this study are listed in Table 3.5 below
Cultivars CU87 CU127 Cuigu Kappa Summer no.7 Kappa Summer no.11
Plant growth and conditions
Each tray contained 108 holes, with one cucumber seedling sown in each hole, using Potgrond H 90 commercial nursery medium (peat: perlite = 9:1) from Klasmann-Deilmann, Germany The cucumber cultivars were then placed in a plant growth chamber that simulated the average winter temperature of central Taiwan, set at 25 ± 1°C during the day and 18 ± 1°C at night, along with a 12-hour photoperiod and light intensity.
70 μmol m -2 s -1 cultured until the plants had two fully expanded leaves for experimental use
Eighteen robust cucumber seedlings from each cultivar were selected for testing and divided into two groups: a chilling group and a control group The chilling group was subjected to a low-temperature environment in a refrigerator, maintained at temperatures of 7 ± 0.5 °C during the day and 4 ± 0.5 °C at night, for a duration of three days This treatment included a photoperiod of 12 hours and a light intensity of 70 μmol m -2 s -1, ensuring continuous low-temperature exposure for the specified period.
The control group and recovery conditions for the chilling injury were maintained at 25/18°C (day/night) during plant cultivation Following the low-temperature treatment, the plants were returned to room temperature for a recovery period of three days.
Experimental design
3.5.1 Change of chlorophyll fluorescence (Fv/Fm)
Chlorophyll fluorescence parameters in cucumber leaves were assessed at room temperature under dark conditions using a MINI-PAM photosynthesis yield analyzer (Walz, Effeltrich, Germany) The analysis focused on the area of the second leaf that excluded the veins, and the plants underwent a 30-minute dark adaptation prior to measurement The results, expressed as a percentage, indicated the relative reduction or recovery of chlorophyll fluorescence in the plants.
Chlorophyll fluorescence was calculated by a formula:
The relative reduction of chlorophyll fluorescence (%) (initial Fv/Fm – after chilling Fv/Fm) / initial Fv/Fm × 100%
The relative recovery of chlorophyll fluorescence (%) (after recovery Fv/Fm – after chilling Fv/Fm) / initial Fv/Fm × 100%
The Chlorophyll fluorescence technique is a promising method for quantitatively assessing chlorophyll index (CI) due to its rapid, sensitive, and non-destructive nature, allowing for the detection of injury before visible symptoms appear By utilizing a fluorometer, this technique can estimate quantum yield (Fv/Fm) and measure the rate of rise in chlorophyll fluorescence, with reductions in these parameters indicating potential plant stress.
16 shown to be highly correlated with chilling stress of leaves of various plant species, as well as with chilling stress of leaves
Plants were subjected to a low-temperature treatment for three days, followed by a recovery period at normal temperatures for an additional three days The chilling injury severity of the second true leaf was visually assessed and categorized into five grades based on the extent of cold damage observed This scoring system was applied to both the low-temperature and recovery treatment groups.
Table 3.6 Appearance severity level of cold damage
Grades Severity of cold injury
1 The second true leaf has > 0 and ≤ 5% leaf area appearance damage
2 The second true leaf has > 5 and ≤ 15% leaf area appearance damage
3 The second true leaf has >15 and ≤ 30% leaf area appearance damage
4 The second true leaf has > 30 and ≤ 50% leaf area appearance damage
5 The second true leaf has over 50% leaf area appearance damage
Visual assessments of external damage, including pitting and decay, are commonly used to evaluate chilling injury in plants, despite their subjective nature To enhance the accuracy of these assessments, we aimed to correlate traditional visual injury scoring, based on the method of Chaplin et al (1991), with more objective measurement techniques such as chlorophyll measurements, electrolyte leakage, and chlorophyll fluorescence.
After the plants were treated for 3 days at recovery temperature, samplings with the second true leaf for 10 mm diameter leaf disc; each tube has 6 discs which contain
To measure the electric conductivity of a solution, start by shaking 10 ml of pure water for 3 hours and then measure its electrical conductivity (ECo) Next, treat the sample with hot water at 95 °C for 2 hours and measure the conductivity again (EC1) after allowing it to cool to room temperature These two measurements are then used to calculate the percentage of total electric conductivity of the solution.
The electrical conductivity (EC) was calculated by a formula:
Malondialdehyde content was assessed following the modified method of Mao et al (2007) After reaching the two true leaf stage, seedlings underwent a cold treatment for three days Subsequently, leaf samples weighing 0.25 g were immersed in liquid nitrogen and stored at -80°C in an ultra-low temperature freezer for testing.
Leaf samples (0.25 g) were ground in liquid nitrogen and homogenized with 5 ml of 0.1% trichloroacetic acid (TCA) The mixture was centrifuged at 10,000 × g and 20°C for 5 minutes Next, 1 ml of the supernatant was combined with 4 ml of 0.5% thiobarbituric acid (TBA) prepared with 20% TCA, and the mixture was heated in a 95°C water bath for 15 minutes before being placed in an ice bath immediately Subsequently, samples were centrifuged at 12,000 × g and 4°C for 10 minutes Finally, 0.2 ml of the supernatant was transferred to 96-well plates, and the absorbance was measured at 532 nm using ELISA, with non-specific absorption recorded at 600 nm.
(ELISA) was subtracted The amount of MDA parameter was calculated from the extinction coefficient 155 mM -1 cm -1
The MDA was calculated by following formula:
MDA (nmol g/FW) = [(OD532-OD600)/155 x 5 x 5]/FW x 1000/1
Statistical analysis
Standard error calculations are performed using Microsoft Excel, while data significance is analyzed through analysis of variance (ANOVA) with the Statistical Analysis System (SAS) Differences between data sets are assessed using a T-test at a 5% significance level (P < 0.05) In tables, different letters (a, b, c) indicate significant differences between genotypes at the same temperature.
RESULTS
Chlorophyll fluorescence (Fv/Fm)
To analyze PSII activity in photosynthesis, the Fv/Fm value, calculated as Fv=Fm – Fo, was measured Here, Fo represents the minimum and Fm the maximum chlorophyll fluorescence values of dark-adapted leaves, providing insight into the efficiency of photosystem II.
Chlorophyll fluorescence measurements were conducted to evaluate chilling injury in cucumber (Cucumis sativus L.) leaves, revealing that the changes in the ratios of variable to chlorophyll fluorescence indicate chilling sensitivity rather than membrane damage This highlights the inherent photosynthetic capacity of plants and their response to chilling stress (Li et al 2004).
Figure 4.1 Chlorophyll fluorescence of 6 cucumber cultivars under control
CU87 CU127 Cuigu Kappa summer no 7
C h lo ro p h y ll f lu o re sc en ce ( F v /F m )
The chlorophyll fluorescence parameters in the control group remained consistent from day 0 to recovery day 3, averaging around 0.800 (Fv/Fm) (Figure 4.1) Since this group received no treatment, the parameters remained high with no significant changes observed after 6 days, indicating that the plants continued to thrive under control conditions.
In chilling group, the results showed that chilling condition effects on chlorophyll fluorescence parameters were very serious (Figure 4.2)
Figure 4.2 Chlorophyll fluorescence (Fv/Fm) of 6 cucumber cultivars under 6 chilling days
At day 0, the chlorophyll fluorescence levels of six cultivars were approximately 0.800 However, when subjected to chilling conditions, these parameters rapidly declined over three days, particularly in cultivars CU87 and CU127.
CU87 CU127 Cuigu Kappa summer no 7
C h lo ro p h y ll f lu o re sc e n ce ( F v /F m )
The chlorophyll fluorescence parameter for CU127 was lower than that of other cultivars, with its maximum Fv/Fm value reaching only 0.500, while other cultivars exceeded 0.600 by recovery day 3 Despite a smooth recovery in plant growth after returning to normal conditions, CU127 lagged behind In contrast, both Kappa summer no 7 and Kappa summer no 11 exhibited higher chlorophyll fluorescence levels than the other cultivars following rewarming.
The graph illustrates daily chlorophyll fluorescence data over a six-day period for each cultivar, while Table 4.1 presents specific percentages indicating the reduction and recovery of plants following six days of treatment.
Table 4.1 Data of reduction and recovery of chlorophyll fluorescence (%) of chilling group
Day 1 Day 2 Day 3 Recovery day 1
The result of chlorophyll fluorescence has shown evidence that under chilling conditions, plant growth ceased for 6 cucumber cultivars and restarted after returning to the optimal growth conditions (Figure 4.3); (Figure 4.4)
Figure 4.3 Relative reductions of chlorophyll fluorescence (%) under 3 days chilling; Vertical bars represent the standard deviation of the mean Different letter indicate the significant differences between cultivar at P