J Plant Biotechnol 2017; 44(3): 229-234
Published online September 30, 2017
https://doi.org/10.5010/JPB.2017.44.3.229
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: rizkita@sith.itb.ac.id
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Photoperiod is one of the factors affecting productivity of cucumber plant by inducing ethylene hormone production and so triggering flower sex differentiation into female flower. However, only few studies have been perfomed in order to reveal the effect of photoperiod in molecular level in relation to the flower differentiation. Therefore, in this study, Mercy cultivar of cucumber (
Keywords
Production of cucumber (
Conventional cultivation of some cucumber cultivars resulted a very low productivity due to low numbers of female flowers produced in the early stage of flowering (Johnson 2008). One of the factors that affects flowering is photoperiod. Generally, cucumber is known as a neutral day plant, in which its flowering process is independent of daylength (Savonen 2003; Reece, et al. 2012). However, based on other study,
Other factor that can affect flowering process in cucumber is the production of ethylene, which is affected by the presence of sucrose inside the plant. High sucrose content will correlate to higher production of ethylene (Miao et al. 2010). According to Yamasaki et al (2010), the increase of ethylene in short-day plants is a result of an increase in expression of
Cucumber’s seeds used in this experiment were from Mercy cultivar that represent
Photoperiod used in this experiment represent the short-day (8 h irradiation), neutral-day (12 h irradiation), and long-day plants (16 h irradiation). Photoperiod for short-day treatment was achieved by using 2-ply 75% shade net. Neutral and long day photoperiod were achieved by using white LEDs with Photosynthetic Active Radiation (PAR) of 100 ~ 220 µmol·m-2·s-1 and light intensity 600 ~ 10.000 lux with total power input 74 watt. LEDs light was set to turned on for neutral-day and long-day photoperiod at 5.00 ~ 6.00 PM and 5.00 ~ 10.00 PM, respectively. For control treatment, cucumbers were not given any light treatment. Plants were watered using half liter of tap water each day until 3 weeks, and then watered with a liter/day until harvest. Plants were fertilized with NPK fertilizer (16:16:16). Experiment were conducted in ITB Jatinangor screen house.
Observation and quantification of flowers was conducted manually by the end of the treatments. The flowers that were counted were male and female flowers from main and lateral stems.
Data were analyzed using one way ANOVA and further by using Duncan Test at the level of p ≤ 0,05 using SPSS Software version 22nd.
Table 1 Primers used in PCR reaction
Primers | Nucleotide sequence (5’ < 3’) | Amplicon (bp) |
---|---|---|
CsGaDPH F | TCAACGACCCCTTCATCAC | 236 |
CsGaDPH R | AGCAGCCTTGTCCTTGTCA | |
CsACS2 F | CTCACAAACGCAACGGTTTT | 167 |
CsACS2 R | CTCAAATTCATCGGCATTCCCT | |
CsETR1 F | GTTGTTGCTGTCCGTGTTCC | 670 |
CsETR1 R | ACCCACAACATTAAGAATAGCTTC | |
CsCaN F | TCACGGATGCTGGTTACAGG | 836 |
CsCaN R | TGGTCTTAGGCGGCAAGATT | |
CsPIF4 F | TGTCTCGAATGGCTATGGGC | 533 |
CsPIF4 R | CTTCTACTTGGTGGAGAGCAAGG |
Sampling method was modified based on Yamasaki’s research. Apical and lateral shoot of cucumber were excised and used for total RNA extraction with Promega SV Total RNA Isolation System (Catalog No: Z3100) based on manufacturer’s instructions. Isolated RNAs were cleaned up from DNA contaminant using DNase I Thermo Scientific (Catalog No: #EN0521). Isolated RNA was treated with with Bio-rad iScript™ cDNA Synthesis Kit (Catalog No: 170-8890) to produce First strand cDNA by previously calculating RNA concentration and RNA quality using spectrophotometer at 260, 280, 320, and 230 nm. Synthesized cDNA was used as a template for PCR confirmation with Promega GoTaq Green Master Mix (Catalog No: M7122) and ABI Veriti® thermocycler.
Total male and female flowers produced from photoperiod treatments was shown in Figure 1A. This figure showed that Mercy was cucumber from
(A) Total number of flowers produced by Mercy cultivar. (B) Comparison of female flowers produced by Mercy cultivar in the main stem (light orange) and lateral stem (dark orange)
Another finding in this experiment was that the main and lateral stems produced different numbers of female flowers (Figure 1B). Eight hours irradiation produced more female flowers in the main stem, while 12 and 16 hours irradiation showed more female flowers in their lateral stem. The maximum number of female flowers in the main stem could be explained by the production of ethylene in apical shoot induced by shorter photoperiod. This finding was supported by Yamasaki research that showed the same effect of photoperiod (Yamasaki et al. 2003). High female flowers on cucumber main stem was also related to the increase of ethylene production that induced by shorter photoperiod (Ikram et al. 2015; Yamasaki et al. 2003). Interestingly, prolonged photoperiod to 16 hours significantly increased the female flowers in lateral stem, while the total number of female flowers in the plant was similar compared to 8 hours. Longer photoperiod increased sugar content in cucumber (Mayorazaki et al. 2015). Escalation of sugar content affected the lateral bud development (Mason et al. 2016). Apical dominance known to be affected by the role of auxin, but Mason (2014) also suggested that it may not be controlled solely by auxin, it was also controlled by the needs of sugar from shoot tip. Excised shoot tip’s canceled apical dominance as a result from distributed sugar content to axillary buds (Mason et al. 2014). Therefore, higher sugar content induced by longer photoperiod influenced the development of lateral buds. Increased sugar content affected the production of lateral stem, the development of flowers, and induced ethylene production to stimulate flower differentiation to female flower (Miao et al. 2010; Ikram et al. 2015; Mason et al. 2014).
In spite of its difference in flower differentiation, photoperiod did not play a major role in initiation of flowering, all treatment conditions induced flowering. Therefore, cucumber could be categorized as neutral-day plant. This statement was supported by other research that showed cucumber was into neutral-day plant (Savonen 2003). It is suggested that flower production might be induced by autonomous pathway or sugar content (Taiz and Zeiger 2010; Ikram et al. 2015). In addition, Figure 1 showed that photoperiod on cucumber played a major role in sex determination. This result supported by other research such as Yamasaki (2003), Wang (2010), and Pessarakli (2016). Flower differentiation in Melon (
Probability of cucumber production in Mercy cultivar depends on the capability of cucumber plant to produce female flowers (Pessarakli 2016). Even though the total number of female flowers in 8 hours and 16 hours photoperiod did not differ significantly, the energy consumption to produce each female flowers was different. 16 hours light treatment need an additional light supply in the cultivation, 8 hours photoperiod did not need any additional energy. Hence, the best photoperiod for producing female flower in cucumber Mercy cultivar was 8 hours irradiation.
Result from RNA extraction was shown by electrophoregram in Figure 3A. cDNA was synthesized from the RNA and kept at -20°C. Gene expression analysis to confirm the model was conducted by using
Proposed model of ethylene affecting flower differentiation in cucumber
(A) Electrophoregram for RNA extraction (Note : M = Mercy; 8,12,16 = Photoperiod). (B) Electrophoregram for gene expression analysis related to photoperiod, ethylene, and flower differentiation. (Note : M = Mercy; 8,12,16 = photoperiod; A = ACS; C = CaN; E = ETR; P = PIF4; G = GaDPH; L = Ladder; Black arrow = ACS gene; Orange arrow = CaN gene; Blue arrow = ETR gene; Green arrow: GaDPH gene)
Figure 3B showed the result of Mercy that was given 8 hours and 16 hours light treatments expressed
Ethylene is commonly known as plant hormone that plays important role in fruit ripening, plant growth, maturation, and signalling for self-defense mechanism (Taiz and Zeiger 2010). However, limited study has described the signal transduction pathway of ethylene in the process of sexual differentiation of flowers. Nevertheless there were studies that explained that ethylene was received by ETR receptor in hermaphrodite flowers and the response was transduced to the activation of calcium-dependent nuclease in order to stop the development of primordial anther (Gu et al. 2011; Bai and Xu 2013). Therefore, alternative/additional signal transduction process that can complement the data basis on KEGG to relate phytochrome and ethylene and ethylene with flower sex determination is needed.
Based on results from this research, it was confirmed that
Best photoperiod to achieve optimum cucumber productivity depends on its cultivar, as it based on its sex differences in flowers production. In Mercy cultivar, female flower production affected the productivity more than male flowers. The condition to achieve the highest productivity was 8 hours treatment which produced 14.7 female flowers and 54.7 male flowers.
This proposed model was confirmed by electrophoresis that showed bands of
The researchers would like to thank Lembaga Pengelola Dana Pendidikan (LPDP) for giving research grant and PT. East West Seed Indonesia for providing cucumber’s seeds used in this research.
J Plant Biotechnol 2017; 44(3): 229-234
Published online September 30, 2017 https://doi.org/10.5010/JPB.2017.44.3.229
Copyright © The Korean Society of Plant Biotechnology.
Muhammad Maulana Malikul Ikram, Rizkita Rachmi Esyanti
School of Life Science and Technology, Institut Teknologi Bandung, Bandung 40132, Indonesia
Correspondence to: e-mail: rizkita@sith.itb.ac.id
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Photoperiod is one of the factors affecting productivity of cucumber plant by inducing ethylene hormone production and so triggering flower sex differentiation into female flower. However, only few studies have been perfomed in order to reveal the effect of photoperiod in molecular level in relation to the flower differentiation. Therefore, in this study, Mercy cultivar of cucumber (
Keywords:
Production of cucumber (
Conventional cultivation of some cucumber cultivars resulted a very low productivity due to low numbers of female flowers produced in the early stage of flowering (Johnson 2008). One of the factors that affects flowering is photoperiod. Generally, cucumber is known as a neutral day plant, in which its flowering process is independent of daylength (Savonen 2003; Reece, et al. 2012). However, based on other study,
Other factor that can affect flowering process in cucumber is the production of ethylene, which is affected by the presence of sucrose inside the plant. High sucrose content will correlate to higher production of ethylene (Miao et al. 2010). According to Yamasaki et al (2010), the increase of ethylene in short-day plants is a result of an increase in expression of
Cucumber’s seeds used in this experiment were from Mercy cultivar that represent
Photoperiod used in this experiment represent the short-day (8 h irradiation), neutral-day (12 h irradiation), and long-day plants (16 h irradiation). Photoperiod for short-day treatment was achieved by using 2-ply 75% shade net. Neutral and long day photoperiod were achieved by using white LEDs with Photosynthetic Active Radiation (PAR) of 100 ~ 220 µmol·m-2·s-1 and light intensity 600 ~ 10.000 lux with total power input 74 watt. LEDs light was set to turned on for neutral-day and long-day photoperiod at 5.00 ~ 6.00 PM and 5.00 ~ 10.00 PM, respectively. For control treatment, cucumbers were not given any light treatment. Plants were watered using half liter of tap water each day until 3 weeks, and then watered with a liter/day until harvest. Plants were fertilized with NPK fertilizer (16:16:16). Experiment were conducted in ITB Jatinangor screen house.
Observation and quantification of flowers was conducted manually by the end of the treatments. The flowers that were counted were male and female flowers from main and lateral stems.
Data were analyzed using one way ANOVA and further by using Duncan Test at the level of p ≤ 0,05 using SPSS Software version 22nd.
Table 1 . Primers used in PCR reaction.
Primers | Nucleotide sequence (5’ < 3’) | Amplicon (bp) |
---|---|---|
CsGaDPH F | TCAACGACCCCTTCATCAC | 236 |
CsGaDPH R | AGCAGCCTTGTCCTTGTCA | |
CsACS2 F | CTCACAAACGCAACGGTTTT | 167 |
CsACS2 R | CTCAAATTCATCGGCATTCCCT | |
CsETR1 F | GTTGTTGCTGTCCGTGTTCC | 670 |
CsETR1 R | ACCCACAACATTAAGAATAGCTTC | |
CsCaN F | TCACGGATGCTGGTTACAGG | 836 |
CsCaN R | TGGTCTTAGGCGGCAAGATT | |
CsPIF4 F | TGTCTCGAATGGCTATGGGC | 533 |
CsPIF4 R | CTTCTACTTGGTGGAGAGCAAGG |
Sampling method was modified based on Yamasaki’s research. Apical and lateral shoot of cucumber were excised and used for total RNA extraction with Promega SV Total RNA Isolation System (Catalog No: Z3100) based on manufacturer’s instructions. Isolated RNAs were cleaned up from DNA contaminant using DNase I Thermo Scientific (Catalog No: #EN0521). Isolated RNA was treated with with Bio-rad iScript™ cDNA Synthesis Kit (Catalog No: 170-8890) to produce First strand cDNA by previously calculating RNA concentration and RNA quality using spectrophotometer at 260, 280, 320, and 230 nm. Synthesized cDNA was used as a template for PCR confirmation with Promega GoTaq Green Master Mix (Catalog No: M7122) and ABI Veriti® thermocycler.
Total male and female flowers produced from photoperiod treatments was shown in Figure 1A. This figure showed that Mercy was cucumber from
(A) Total number of flowers produced by Mercy cultivar. (B) Comparison of female flowers produced by Mercy cultivar in the main stem (light orange) and lateral stem (dark orange)
Another finding in this experiment was that the main and lateral stems produced different numbers of female flowers (Figure 1B). Eight hours irradiation produced more female flowers in the main stem, while 12 and 16 hours irradiation showed more female flowers in their lateral stem. The maximum number of female flowers in the main stem could be explained by the production of ethylene in apical shoot induced by shorter photoperiod. This finding was supported by Yamasaki research that showed the same effect of photoperiod (Yamasaki et al. 2003). High female flowers on cucumber main stem was also related to the increase of ethylene production that induced by shorter photoperiod (Ikram et al. 2015; Yamasaki et al. 2003). Interestingly, prolonged photoperiod to 16 hours significantly increased the female flowers in lateral stem, while the total number of female flowers in the plant was similar compared to 8 hours. Longer photoperiod increased sugar content in cucumber (Mayorazaki et al. 2015). Escalation of sugar content affected the lateral bud development (Mason et al. 2016). Apical dominance known to be affected by the role of auxin, but Mason (2014) also suggested that it may not be controlled solely by auxin, it was also controlled by the needs of sugar from shoot tip. Excised shoot tip’s canceled apical dominance as a result from distributed sugar content to axillary buds (Mason et al. 2014). Therefore, higher sugar content induced by longer photoperiod influenced the development of lateral buds. Increased sugar content affected the production of lateral stem, the development of flowers, and induced ethylene production to stimulate flower differentiation to female flower (Miao et al. 2010; Ikram et al. 2015; Mason et al. 2014).
In spite of its difference in flower differentiation, photoperiod did not play a major role in initiation of flowering, all treatment conditions induced flowering. Therefore, cucumber could be categorized as neutral-day plant. This statement was supported by other research that showed cucumber was into neutral-day plant (Savonen 2003). It is suggested that flower production might be induced by autonomous pathway or sugar content (Taiz and Zeiger 2010; Ikram et al. 2015). In addition, Figure 1 showed that photoperiod on cucumber played a major role in sex determination. This result supported by other research such as Yamasaki (2003), Wang (2010), and Pessarakli (2016). Flower differentiation in Melon (
Probability of cucumber production in Mercy cultivar depends on the capability of cucumber plant to produce female flowers (Pessarakli 2016). Even though the total number of female flowers in 8 hours and 16 hours photoperiod did not differ significantly, the energy consumption to produce each female flowers was different. 16 hours light treatment need an additional light supply in the cultivation, 8 hours photoperiod did not need any additional energy. Hence, the best photoperiod for producing female flower in cucumber Mercy cultivar was 8 hours irradiation.
Result from RNA extraction was shown by electrophoregram in Figure 3A. cDNA was synthesized from the RNA and kept at -20°C. Gene expression analysis to confirm the model was conducted by using
Proposed model of ethylene affecting flower differentiation in cucumber
(A) Electrophoregram for RNA extraction (Note : M = Mercy; 8,12,16 = Photoperiod). (B) Electrophoregram for gene expression analysis related to photoperiod, ethylene, and flower differentiation. (Note : M = Mercy; 8,12,16 = photoperiod; A = ACS; C = CaN; E = ETR; P = PIF4; G = GaDPH; L = Ladder; Black arrow = ACS gene; Orange arrow = CaN gene; Blue arrow = ETR gene; Green arrow: GaDPH gene)
Figure 3B showed the result of Mercy that was given 8 hours and 16 hours light treatments expressed
Ethylene is commonly known as plant hormone that plays important role in fruit ripening, plant growth, maturation, and signalling for self-defense mechanism (Taiz and Zeiger 2010). However, limited study has described the signal transduction pathway of ethylene in the process of sexual differentiation of flowers. Nevertheless there were studies that explained that ethylene was received by ETR receptor in hermaphrodite flowers and the response was transduced to the activation of calcium-dependent nuclease in order to stop the development of primordial anther (Gu et al. 2011; Bai and Xu 2013). Therefore, alternative/additional signal transduction process that can complement the data basis on KEGG to relate phytochrome and ethylene and ethylene with flower sex determination is needed.
Based on results from this research, it was confirmed that
Best photoperiod to achieve optimum cucumber productivity depends on its cultivar, as it based on its sex differences in flowers production. In Mercy cultivar, female flower production affected the productivity more than male flowers. The condition to achieve the highest productivity was 8 hours treatment which produced 14.7 female flowers and 54.7 male flowers.
This proposed model was confirmed by electrophoresis that showed bands of
The researchers would like to thank Lembaga Pengelola Dana Pendidikan (LPDP) for giving research grant and PT. East West Seed Indonesia for providing cucumber’s seeds used in this research.
(A) Total number of flowers produced by Mercy cultivar. (B) Comparison of female flowers produced by Mercy cultivar in the main stem (light orange) and lateral stem (dark orange)
Proposed model of ethylene affecting flower differentiation in cucumber
(A) Electrophoregram for RNA extraction (Note : M = Mercy; 8,12,16 = Photoperiod). (B) Electrophoregram for gene expression analysis related to photoperiod, ethylene, and flower differentiation. (Note : M = Mercy; 8,12,16 = photoperiod; A = ACS; C = CaN; E = ETR; P = PIF4; G = GaDPH; L = Ladder; Black arrow = ACS gene; Orange arrow = CaN gene; Blue arrow = ETR gene; Green arrow: GaDPH gene)
Table 1 . Primers used in PCR reaction.
Primers | Nucleotide sequence (5’ < 3’) | Amplicon (bp) |
---|---|---|
CsGaDPH F | TCAACGACCCCTTCATCAC | 236 |
CsGaDPH R | AGCAGCCTTGTCCTTGTCA | |
CsACS2 F | CTCACAAACGCAACGGTTTT | 167 |
CsACS2 R | CTCAAATTCATCGGCATTCCCT | |
CsETR1 F | GTTGTTGCTGTCCGTGTTCC | 670 |
CsETR1 R | ACCCACAACATTAAGAATAGCTTC | |
CsCaN F | TCACGGATGCTGGTTACAGG | 836 |
CsCaN R | TGGTCTTAGGCGGCAAGATT | |
CsPIF4 F | TGTCTCGAATGGCTATGGGC | 533 |
CsPIF4 R | CTTCTACTTGGTGGAGAGCAAGG |
Journal of
Plant Biotechnology(A) Total number of flowers produced by Mercy cultivar. (B) Comparison of female flowers produced by Mercy cultivar in the main stem (light orange) and lateral stem (dark orange)
|@|~(^,^)~|@|Proposed model of ethylene affecting flower differentiation in cucumber
|@|~(^,^)~|@|(A) Electrophoregram for RNA extraction (Note : M = Mercy; 8,12,16 = Photoperiod). (B) Electrophoregram for gene expression analysis related to photoperiod, ethylene, and flower differentiation. (Note : M = Mercy; 8,12,16 = photoperiod; A = ACS; C = CaN; E = ETR; P = PIF4; G = GaDPH; L = Ladder; Black arrow = ACS gene; Orange arrow = CaN gene; Blue arrow = ETR gene; Green arrow: GaDPH gene)