INTRODUCTION
Research rationale
Climate change has significantly impacted the human environment, nature, and wildlife, with agricultural plants being particularly vulnerable These plants are crucial to the economies of many countries and global trade, making them sensitive to weather fluctuations Variations in rainfall, temperature extremes, and severe weather events can drastically affect local crop yields Unexpected climate changes pose serious threats to agricultural production, as temperature fluctuations disrupt cellular physiology in plants Low temperatures, specifically chilling stress (1–10°C), can lead to oxidative stress by creating 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.), which thrive at temperatures above 20°C, face significant challenges in seedling establishment and production due to frost sensitivity Chilling injury, influenced by air temperature, duration of exposure, and plant growth stage, often becomes evident when plants are moved to controlled temperatures This condition leads to the degradation of membrane lipids in the leaves of 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 Taiwan's greenhouses during winter, particularly affecting chilling-sensitive plants like cucumbers These chilling temperatures lead to slowed growth, delayed development, and an extended growing season, especially in susceptible species (Ting et al 1991; Skrudlik and Koscielniak, 1996) Consequently, cucumber growth is hindered, resulting in fewer newly formed plant organs, altered root structures, and decreased rates of flowering, fruiting, and seed filling (Buis et al 1988) This research 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 investigate how low temperatures affect the growth of cucumber seedlings, providing valuable insights for farmers in selecting 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 certain limitations, including a relatively short internship period and the lengthy duration required to complete each experiment focused on plant growth Additionally, the results were not entirely precise, as various cucumber cultivars exhibit different growth rates and levels of environmental adaptation.
Definitions
Chilling temperatures in temperate climates can lead to reduced crop yields or complete failures due to direct damage or delayed maturation Cucumber cultivars, in particular, are susceptible to chilling injury, which affects chilling-sensitive plant species that struggle to withstand low temperatures Cold resistance refers to the ability of these plants to survive chilling conditions during their vegetative state without hindering 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 key physiological processes, including water management, mineral nutrition, photosynthesis, respiration, and overall metabolism (Lukatkin et al 2012) This research specifically aims to investigate the effects of these disturbances on photosynthesis.
Photosynthesis is highly sensitive to cold stress, leading to reduced growth and productivity in plants at low temperatures This complex process involves various components, including CO2 reduction and photosynthetic photosystems, which are crucial for plant health and development.
Photosystem II is identified as the most cold-sensitive component of the electron transport system, as chilling stress notably reduces the maximum PSII quantum yield (Fv/Fm) This decrease in Fv/Fm indicates damage to photochemical reactions and disrupts electron transport by inhibiting electron transfer.
Photosystems are essential protein complexes that play a crucial role in photosynthesis by facilitating the absorption of light and the transfer of energy and electrons These functional and structural units are located in the thylakoid membranes of chloroplasts in plants and in the cytoplasmic membranes of photosynthetic bacteria.
Photosystem II (PSII) is the initial protein complex in the light-dependent reactions of oxygenic photosynthesis, found in the thylakoid membrane of plants It harnesses photons of light to energize electrons, which are then transferred through various coenzymes and cofactors, reducing plastoquinone to plastoquinol This process involves oxidizing water, resulting in the production of hydrogen ions and molecular oxygen By replenishing lost electrons through the splitting of water, PSII plays a crucial role in facilitating the entire photosynthesis process.
Chilling-sensitive plants experience a significant reduction in photosynthesis rates during and after chilling periods, primarily due to lower temperatures and prolonged chilling durations This decrease can persist even after the plants are moved back to warmer conditions (Kingston et al 1999) The suppression of photosynthesis is attributed to several physiological factors, including the inhibition of carbohydrate transport via phloem, limitations in stomatal function, and damage to the photosynthetic machinery.
The water-splitting complex of photosystem II produces superoxide (O2 -), a reactive oxygen species (ROS) that inhibits electron transport and disrupts energy storage, affecting the synthesis of key enzymes in the Calvin and C4 cycles Cold-sensitive plant species exhibit a narrower temperature equilibrium for leaf photosynthesis compared to cold-tolerant species (Yamori et al 2009) Chilling sensitive plants under light significantly impacts their photosynthetic apparatus more than chilling in darkness (Szalai et al 2006) Photoinhibition, characterized by reduced photosynthetic activity due to excessive light during chilling, intensifies with lower temperatures and higher light intensity (Greer, 1995) While the primary site of photoinhibition is photosystem II, it has been found that damage can also occur at photosystem I under relatively low light and temperature conditions (Sonoike).
Chilling temperatures can lead to a decrease in photosynthesis due to photo-oxidative damage to chloroplast membranes, resulting in 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 a reduction in the antioxidant activity of plant tissues.
Cell membrane changes
Low temperatures significantly affect the physical properties of plant cell membranes, particularly in sensitive species Chilling stress reduces membrane elasticity and lipid inclusion, which in turn decreases lipid fluidity This reduction adversely impacts the activity of membrane-bound enzymes, such as H+-ATPase, while also increasing the lateral diffusion of phospholipids.
Chilling injury occurs when low temperatures directly affect cellular components, leading to alterations in membrane characteristics and enzyme conformations (Quinn, 1988; Kasamo et al., 1992; Koster et al., 1994; Kasamo et al., 2000) These effects can be reversed if the plant is quickly moved to warmer conditions before visible damage occurs However, prolonged exposure to cold results in significant membrane deterioration, loss of integrity, and increased electrolyte leakage, ultimately decreasing mitochondrial oxidative activity and photosynthesis rates This disruption leads to metabolic imbalances, toxic substance accumulation, and the visible symptoms of chilling injury (Parkin et al., 1989; 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, which compromises membrane integrity and cell compartmentation in chilled conditions Elevated solute and electrolyte leakage is observed in various chilled tissues and serves as an indicator of membrane damage This leakage can be attributed to the formation of membrane domains with differing configurations due to exposure to cold temperatures.
11 induced changes in lipid phases (Leshem, 1992), or from damage of membrane, particularly as regards lipids (Harwood, 2005)
Malondialdehyde is a key indicator of oxidative damage in cells due to stress-induced lipid peroxidation of polyunsaturated fatty acids Environmental stress, particularly low temperatures, disrupts the metabolism of active oxygen species, leading to their accumulation and the degradation of essential scavenging enzymes like SOD, CAT, POD, and APX Various stress factors are known to enhance the production of reactive oxygen species (ROS) within plant cells, primarily originating from the photosynthetic electron transport chain This ROS generation is intensified under environmental stress, resulting in the downregulation of photosynthetic dark reactions and an over-reduction of the electron transport chain, ultimately producing harmful ROS such as superoxide (O2 -) and hydrogen peroxide (H2O2 -).
Under chilling conditions, the accumulation of reactive oxygen species (ROS), such as superoxide (O2 -) and hydrogen peroxide (H2O2 -), is linked to chloroplast damage While a low level of ROS is necessary for plant functions, excessive accumulation can result in lipid peroxidation, protein oxidation, and DNA damage This lipid peroxidation causes structural abnormalities and dysfunction in cells, highlighting the delicate balance required for ROS in plant health.
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 hole in the tray was filled with one cucumber seedling, totaling 108 holes per tray, 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 to mimic the average winter temperatures of central Taiwan, set at 25 ± 1 °C during the day and 18 ± 1 °C at night, with a photoperiod of 12 hours of light.
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 throughout the testing 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 and under dark conditions using a MINI-PAM photosynthesis yield analyzer (Walz, Effeltrich, Germany) The analysis focused on the leaf area excluding the veins, with plants undergoing a 30-minute dark adaptation prior to measurement The results, calculated using specific formulas, were expressed as a percentage representing 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 the quantitative assessment of chlorophyll integrity (CI) due to its rapid, sensitive, and non-destructive nature, allowing for the detection of injury before visible symptoms appear By utilizing a fluorometer, researchers can estimate quantum yield (Fv/Fm) and measure the rise rate in chlorophyll fluorescence, with reductions in these parameters indicating potential plant stress or damage.
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 low temperatures for three days, followed by a recovery period at normal temperatures for an additional three days The chilling injury of the second true leaf was assessed visually, categorizing the cold damage into five grades for 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 visual injury scores with objective measurement techniques such as chlorophyll measurements, electrolyte leakage, and chlorophyll fluorescence, following the scoring method established by Chaplin et al (1991).
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 determine the total electric conductivity of a sample, 10 ml of pure water is shaken for 3 hours before measuring its electrical conductivity (ECo) The sample is then subjected to a hot water bath at 95 °C for 2 hours, followed by a measurement of the conductivity (EC1) after cooling to room temperature These two measurements are utilized to calculate the percentage of the total electric conductivity of the solution.
The electrical conductivity (EC) was calculated by a formula:
Malondialdehyde levels were assessed following the modified method of Mao et al (2007) After reaching the two true leaf stage, seedlings underwent a 3-day cold treatment, after which leaf samples weighing 0.25 g were immersed in liquid nitrogen and stored at -80°C 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 Subsequently, 1 ml of the supernatant was combined with 4 ml of 0.5% thiobarbituric acid (TBA), prepared with 20% TCA, and heated in a 95 °C water bath for 15 minutes before being placed in an ice bath The samples were then 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 absorption 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 among genotypes at the same temperature.
RESULTS
Chlorophyll fluorescence (Fv/Fm)
To analyze PSII activity in photosynthesis, the Fv/Fm value was measured, where Fv represents the difference between the maximum (Fm) and minimum (Fo) chlorophyll fluorescence of dark-adapted leaves.
Chlorophyll fluorescence measurements were utilized to evaluate chilling injury in cucumber (Cucumis sativus L.) leaves, revealing that the chilling-dependent changes in the variable to chlorophyll fluorescence ratios indicated chilling sensitivity rather than membrane damage, highlighting the inherent photosynthetic capacity of plants (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), as shown in Figure 4.1 Due to the absence of treatment, these parameters remained elevated, exhibiting no significant changes over six days, while the plants continued to thrive under controlled 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 of six cultivars was approximately 0.800 However, when subjected to chilling conditions, the fluorescence levels rapidly decreased 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 )
Chlorophyll fluorescence parameters were lower in the CU127 cultivar compared to others While most plants showed smooth recovery in growth after returning to normal conditions, CU127 only achieved a maximum Fv/Fm of 0.500, whereas other cultivars exceeded 0.600 by day 3 of recovery Notably, 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 provides detailed percentages of plant reduction and recovery 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