Effect on growth of cucumber seedlings at low temperature

In this study, series of experiments about cucumber seedlings treated with low temperature were tested to see whether their chilling tolerance would be significantly affected; and compar

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

Study mode: Full-time

Major: Environmental Science and Management

Faculty: Advanced Education Program Office

Batch: 2014 - 2018

Thai Nguyen, 16/08/2018

Thai Nguyen University Of Agriculture And Forestry

Degree Program: Bachelor of Environmental Science and Management

Low temperature stresses are major environmental limiters for horticultural production

in greenhouses in Taiwan during winter period The exposure of tropical and

subtropical plants such as cucumber (Cucumis sativus L.) to low temperatures

summer no.7, Kappa summer no.11, CU-74) were cultivated from seed to the seedlings which have two true leaves for testing in control conditions (25/18˚C, day/night), chilling treatment (7/4˚C, day/night) and upon re-warming to (25/18˚C, day/night) In this study, series of experiments about cucumber seedlings treated with low temperature were tested to see whether their chilling tolerance would be significantly affected; and compare the effects of a cold treatment on membrane integrity of six cucumber cultivars and their recovery The results showed that cucumbers were sensitive to chilling stresses, which made the leaves wither in some cucumber seedlings and inhibited cucumber growth in this study Finally, Kappa summer no.7 exhibited as the highest cold resistance cultivar which is suitable for using in winter weather

Kappa summer no.7, cold resistance

Date of submission: 16/08/2018

I would like to express my deep and sincere gratitude to Prof San Gwang

Hwang for the continuous support of my internship study and research He always

helped me any time and also gave me the best conditions, supported all materials for my research and discussed any problems I got whenever I did experiment in his Vegetable laboratory

Special thanks must go to Dr Duong Van Thao who always supported and

cheered me up whole the time He gave me valuable and constructive comments on the manuscript and helped me to increase the quality of the thesis

I wish to express my sincere thanks to Wayne Shih- a master student who

helped me a lot for my experiment during the time I worked with him Without his assistance and dedicated involvement in every step throughout the process, this paper would have never been accomplished

I would like to thank the lab mates for their help and sharing during the time I

who have been studying in NCHU for their sharing to overcome all obstructs in the study-time, we have had useful discussions for my research

Finally I deeply thank my parents, who supported me with love and understanding It was their love that raised me up again when I got weary Without you, I could never have reached this current level of success

Sincerely,

Nguyen Ha Trang

TABLE OF CONTENT

LIST OF FIGURES vii

LIST OF TABLES viii

LIST OF ABBREVIATIONS ix

PART I INTRODUCTION 1

1.1 Research rationale 1

1.2 Research’s objectives 3

1.3 Research questions and hypotheses 3

1.4 Limitations 3

1.5 Definitions 4

PART II LITERATURE REVIEW 5

2.1 An overview about six cucumber cultivars 6

2.2 Effect of chilling on the physiological processes in chilling-sensitive plants 7

2.3 Cell membrane changes 9

PART III METHODS 12

3.1 List of instruments 12

3.2 List of chemicals 12

3.3 Materials 14

3.4 Plant growth and conditions 14

3.5 Experimental design 15

3.5.1 Change of chlorophyll fluorescence 15

3.5.4 MDA content measurement 17

3.6 Statistical analysis 18

PART IV RESULTS 19

4.1 Chlorophyll fluorescence (Fv/Fm) 19

4.2 Severity of chilling injury 23

4.3 Leaf electrolyte leakage 28

4.4 Malondialdehyde 29

PART V DISCUSSION AND CONCLUSION 31

REFERENCES 34

LIST OF FIGURES

Figure 4.1 Chlorophyll fluorescence of 6 cultivars of cucumber under control 19 Figure 4.2 Chlorophyll fluorescence of 6 cultivars of cucumber under 6 days

chilling 20 Figure 4.3 Relative reductions of chlorophyll fluorescence (%) under 3 days

chilling 22 Figure 4.4 Relative recovery of chlorophyll fluorescence (%) after 3 days chilling 23 Figure 4.5 Five levels severity of cold injury of the leaves 25 Figure 4.6 Control group at (a) day 0, (b) recovery day 3 26 Figure 4.7 The chilling group at (a) day 0, (b) recovery day 3 27 Figure 4.8 The severity of chilling injury between first leaf and second leaf of six cultivars of chilling group 27 Figure 4.9 Leaf electrolyte leakage (%) of 6 cucumber cultivars under chilling 29 Figure 4.10 Malondialdehyde (nmol g/FW) of 6 cucumber cultivars under chilling 30

LIST OF TABLES

Table 2.1 List of vegetables, sensitive to chilling temperatures, the lowest safe

storage temperature and the symptoms of chilling injury 5

Table 2.2 Six cucumber cultivars information 7

Table 3.1 Name and commercial company of all instruments used in this study 12

Table 3.2 Properties of liquid nitrogen 12

Table 3.3 Properties of trichloroacetic acid 13

Table 3.4 Properties of thiobarbituric acid 13

Table 3.5 Six cucumber cultivars 14

Table 3.6 Appearance severity level of cold damage 16

Table 4.1 Data of reduction and recovery of chlorophyll fluorescence (%) of chilling group 21

ROS Reactive oxygen species

SCI Severity of cold injury

PART I INTRODUCTION

1.1 Research rationale

In recent years, climate change has shown global impacts on the human environment, nature, wildlife The most vulnerable to climate change is agricultural plants that are one of society's key sensitivities to climate Many agricultural plants, main economic source of some countries and large sectors of world trade, are extremely susceptible to weather changes Small or huge amount of rainfall, a hot spell

or cold snap at the wrong time, or extremes, like flooding and storms, may cause a significant effect on local crop yields Unexpected climate changes can affect production of these plants in the most disastrous way Temperature is continually changed due to climate change and has become a serious threat to crop yield As sessile organisms, plants are constantly exposed to change in temperature and other abiotic factors Low temperatures stress or chilling temperature (1–10°C) is one of the majors abiotic stresses that disturbs cellular physiology, causing oxidation stress via creating imbalance between generation and metabolism of reactive oxygen species leading finally to cell and plant death (Lukatkin and Anjum 2014)

The exposure of tropical and subtropical plants such as cucumber (Cucumis

sativus L.) - a frost-sensitive plant species grows best at temperatures above 20°C and

constitutes a severe limitation to their seedling establishment, physiology and production at low temperatures The magnitude of chilling injury depends on air temperature, time of exposition, plant growth stage and it usually manifests itself after plants are transferred to control temperature Chilling temperatures in leaves of sensitive plants cause the degradation of membrane lipids and decrease the integrity of

cell membranes which change the composition of photosynthetic pigments, decrease leaf stomatal conductance and photosynthesis The reduction in CO2 fixation rate induced by low-temperature stress leads to excessive accumulation of reactive oxygen forms and increased activity of antioxidant enzymes

Low-temperature stresses are major environmental limiters for horticultural production in greenhouses in Taiwan during the winter period A characteristic effect

of chilling temperatures on chilling-sensitive plants is growth slowing, more pronounced in susceptible species and varieties in comparison with the tolerant species (Ting et al 1991) In addition, there is a delayed development and lengthening of the growing season (Skrudlik and Koscielniak, 1996) At the same time, the cucumber growth is delayed, reducing the number of newly formed plant organs and the rate of their occurrence, the structure of roots is changed, and flowering rate, fruit rate and seed filling are reduced (Buis et al 1988) This thesis sheds light on the determinant processes involved in the depletion of cucumber seedlings by chilling injuries, revealing that low temperatures have persistent and detrimental effects on cucumber cultivars Moreover, this study showed that exposure of chilling sensitive plants to low temperature causes disturbances in all physiological processes-water regimes, mineral

nutrition, photosynthesis, respiration and metabolism

1.2 Research’s objectives

The main purpose of this study is to identify the effect of low temperature on growth of cucumber seedlings Moreover, this study may help farmers to choose which cucumber cultivars are suitable for using in the winter cultivation

1.3 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?

• Alternative hypothesis:

1 The cucumber seedlings are affected by chilling treatment

2 There are differences between six cucumber cultivars on the cold resistance

• Null hypothesis:

1 The cucumber seedlings are not affected by chilling treatment

2 There are no differences between six cucumber cultivars on the cold resistance

1.4 Limitations

There are some limitations appeared in this study such as the internship period was pretty short and all experiments were based on the plant’s growing, therefore it totally took a long time to complete each individual experiment In addition, the results were not 100% exactly because different cucumber cultivars have different growth rate and environmental adaptation

1.5 Definitions

Chilling temperatures effects on plants in temperate climates leading to a reduction or complete crop failure due to either direct damage or delayed maturation Most of cucumber cultivars are damaged during chilling temperature This damage is

called chilling injury which damages chilling-sensitive plant species

Chilling-sensitive plants are the plants that are Chilling-sensitive to chilling and easily to be damaged at chilling temperatures The ability of plants in a vegetative state to survive the action of chilling temperatures without harm to the future growth and development are called

cold resistance (Lukatkin et al 2012)

PART II LITERATURE REVIEW

Cucumber (Cucumis sativus L.), is a member of the Cucurbitaceae, which

comprises 90 genera and 750 species It is one of the oldest cultivated vegetable crops and is cultivated in nearly all countries of temperature zones (Tatlioglu, 1993) It is a frost-sensitive plant species, growing best at temperatures above 20°C (Table 2.1) Hundreds of cultivars of varying size and color are now grown in warm areas worldwide, commercially and in home gardens A characteristic effect of chilling temperatures on chilling-sensitive plants is growth slowing, more pronounced in susceptible species and varieties of comparison with the tolerant species

Table 2.1 List of vegetables, sensitive to chilling temperatures, the lowest safe

storage temperature and the symptoms of chilling injury (Lukatkin et al 2012)

Crop Lowest safe temperature

(°C)

Chilling injury symptoms

Crop Lowest safe temperature

green)

13

Poor color when ripe, alternaria rot

2.1 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 (%)

Country of origin

Fruit length (cm) Characteristics

High yield, low temperature resistance

strong branching Kappa summer

in the first stalk

2.2 Effect of chilling on the physiological processes in chilling-sensitive plants

Incubation of chilling-sensitive plants at low temperatures induces disturbances

in physiological processes: water regime, mineral nutrition, photosynthesis, respiration and total metabolism (Lukatkin et al 2012) However, photosynthesis is focus to deeply understand the goal of this research

Among many physiological processes, photosynthesis is one of the most sensitive to cold stress, which is the main reason for the reduction or cessation of growth and decreases in productivity of plants at low temperatures Photosynthesis includes numerous components such as CO2 reduction, photosynthetic photosystems

and the electron transport system Among these, photosystem II has been described as the most cold-sensitive This finding has been supported by chilling stress significantly decreased the maximum PSII quantum yield (Fv/Fm), the Fv/Fm decrease reflects the damage to photochemical reactions, accompanied by the interdict of electron transport (inhibition of electron transfer)

Photosystems are functional and structural units of protein complexes involved

in photosynthesis that together carry out the primary photochemistry of photosynthesis: the absorption of light and the transfer of energy and electrons Photosystems are found in the thylakoid membranes of plants which were located in the chloroplasts of plants and in the cytoplasmic membrane of photosynthetic bacteria

PSII: is the first protein complex in the light-dependent reactions of oxygenic photosynthesis that is located in the thylakoid membrane of plants Within the photosystem, enzymes take photons of light to energize electrons that are then transferred through a variety of coenzymes and cofactors to diminish plastoquinone to plastoquinol The energized electrons are changed by oxidizing water to form hydrogen ions and molecular oxygen By replenishing lost electrons with electrons from the splitting of water, photosystem II offers the electrons for all of the photosynthesis to occur

During and after chilling, the rate of photosynthesis in the leaves of sensitive plants decreased and this is more related to decreasing temperature and lengthening of chilling period and persisted for a long time after transfer of chilled plants in the heat (Kingston et al 1999) The physiological reasons for the suppression

chilling-water-splitting complex of photosystem II and superoxide (O2.-) rather than O2 is

considered as one kind of ROS, inhibiting electron transport, and uncoupling of electron transfer and energy storage, alters in the activity and inhibition of synthesis of major enzymes of the Calvin cycle and C4 cycle Cold-sensitive plant species have smaller temperature equilibrium of leaf photosynthesis than cold-tolerant plant species (Yamori et al 2009) Chilling of sensitive plants in light had much stronger effects on the photosynthetic apparatus than chilling in the dark (Szalai et al 2006) Photo inhibition of photosynthesis is the lowering of photosynthetic activity under excessive illumination during chilling (Nie et al 2008) It increases with decreasing temperature and increasing light intensity (Greer, 1995) Primary site photo inhibition is the photosystem II However, it was discovered that photo inhibition occurs at relatively low light and low temperature, and the main site of damage is photosystem I (Sonoike, 1999) Decrease of photosynthesis at chilling temperatures may be a consequence of photo-oxidative damage to the photosystems in the membranes of chloroplasts, which

is manifested by increasing lipid peroxidation, degradation of chlorophyll, carotene, and xanthophyll’s (Fryer et al 1998) It was caused by activated oxygen species and was associated with reduced antioxidant activity of tissues (Leipner et al 1997), (Leipner et al 2008)

2.3 Cell membrane changes

Low temperatures change the physical properties of cell membranes in plants Chilling of sensitive plants leads to various changes in their membranes, especially is reduce the membrane elasticity, decreasing theirs preventing lipid inclusion in their composition, lower lipid fluidity, thereby reducing the activity of several membrane-bound enzymes, including H+-ATPase, increase the lateral diffusion of phospholipids,

sterols and proteins in the plasma membrane (Quinn, 1988), (Kasamo et al 1992), (Koster et al 1994), (Kasamo et al 2000) Chilling injury is a direct effect of low temperature on cellular constituents, which results some changes in membrane features and/or conformational changes in enzymes and structural proteins (Parkin et al 1989) Such direct effects are readily reversible if the plant material is transferred to non-chilling temperatures after a short exposure to chilling and before visible injury is apparent Therefore, it has been suggested that membrane deterioration is a primary factor in both the physiological and visible manifestations of chilling injury After a long chilling period, these changes lead to loss of membrane integrity and compartmentation, the electrolyte leakage, decrease of oxidative activity of mitochondria, increase of the activation energy of membrane-bound enzymes, reduce the rate of photosynthesis, cause disruption and imbalance of metabolism, the accumulation of toxic substances and 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, 1992) Chilling impairments mainly consist of alteration of metabolic processes decrease in enzymatic activities, reduction of photosynthetic capacity and changes in membrane fluidity among others (Dubey, 1997) Such changes are regularly associated with an increase in membrane permeability, affecting membrane integrity and cell compartmentation under chilling conditions Increased rates of solute and electrolyte leakage occur in a variety of chilled tissues and have been used to evaluate membrane damage following chilling (Wright and Simon, 1973) Leakage points may result from

induced changes in lipid phases (Leshem, 1992), or from damage of membrane, particularly as regards lipids (Harwood, 2005)

Malondialdehyde is one of the final products of stress-induced lipid peroxidation of polyunsaturated fatty acids (Leshem, 1987) and often used as an index

of cell oxidative damage under environmental stress (Shen, 1997) Low temperature disrupts the balance of active oxygen species metabolism, leading to their accumulation and destruction of scavenging enzymes such as SOD, CAT, POD and APX (Kang and Saltveit, 2002) A general characteristic of various stress factors is their capability to increase the generation of reactive oxygen species (ROS) in the cells In plants, the photosynthetic electron transport is also the main origin of ROS Indeed, the production of ROS is unavoidable when the photosynthetic electron transport chain operates under aerobic conditions The generation of ROS in photosynthesizing tissues is significantly exacerbated under environmental stress conditions Under these circumstances, the photosynthetic dark reactions are directly

or indirectly down regulated, which results in over reduction of the photosynthetic electron transport chain, photo reduction of di-oxygen and generation of ROS including O2.- and H2O2.- and OH.- (Hajiboland, 2014)

Accumulation of ROS such as O2.- and H2O2.- occurred under chilling and was

associated with signs of chloroplast damage Although low accumulation of ROS is indispensable, excessive accumulations had been shown to be associated with damage from lipid peroxidation, and oxidation of protein and DNA Lipid peroxidation from excessive ROS leads to structural abnormalities and cell dysfunction (Gill and Tuteja, 2010)

PART III METHODS

3.1 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

Taichung, Taiwan Chlorophyll Fluorometer, Mini-Pam, Walz,

High-Speed Refrigerated Centrifuge, CR

3.2 List of chemicals

All chemicals used in this study are shown below

Table 3.2 Properties of liquid nitrogen

Table 3.3 Properties of trichloroacetic acid (TCA)

Molecular formula

Table 3.4 Properties of thiobarbituric acid

Molecular formula

3.3 Materials

All cucumber cultivars used in this study are listed in Table 3.5 below

Table 3.5 Six cucumber cultivars

Cultivars CU87 CU127 Cuigu Kappa Summer no.7 Kappa Summer no.11

CU74

3.4 Plant growth and conditions

One cucumber seedlings were sowed in each hole and 108 holes fully filled per tray as one replication that used Potgrond H 90 commercial nursery medium (peat: perlite = 9:1) from Klasmann-Deilmann, Germany Cucumber cultivars were placed in

a plant growth chamber to simulate the average winter temperature in central Taiwan

of [25 ± 1/18 ± 1 °C (day/night)] and photoperiod At 12 hours, the light intensity was

70 μmol m-2 s-1 cultured until the plants had two fully expanded leaves for experimental use

After that 18 cucumber seedlings of each cultivar which were strong plants were chosen for testing They were divided into 2 groups that are chilling group and control group The chilling groups were placed in a low-temperature refrigerator equipped with a light source at a temperature of [7 ± 0.5 /4 ± 0.5 °C (day/night)] in 3