Abstract: This study was aimed at evaluating the effect of different calcium formulas with various concentration rates on leaf color, contents of phenolic compounds and quinone, Polyphenol oxidase (PPO) activity and browning percentage in Grand Rapids lettuce. A Factorial in Completely Randomized Design was arranged with four replications and composed of two factors: three forms of calcium in terms of calcium chloride (CaCl2), calcium nitrate [(Ca(NO3)2] or calcium oxide (CaO) by soil dressing application with five concentrations (0, 0.5, 1.0, 1.5 or 2.0%). The results showed that plants-treated with 1.5% CaCl2 had the maximal leaf color in term of greenness (a*) values, while treatment of 2% CaCl2 had the lowest contents of phenolic compounds, quinone and activity of Polyphenol oxidase (PPO) which corresponded to the least browning level at harvesting stage.
INTRODUCTION
Today, there is a growing trend among those concerned about people health to consume the valuable nutrition offered by natural fresh vegetables. Lettuce (Lactuca sataiva) is a popular vegetable that can be eaten fresh like fast food or in prepared meals. It is composed of high nutritional benefits especially fiber, calcium, vitamin C, vitamin A and beta-carotene which functions as an important antioxidant (Lister, 2003). In addition, dietary antioxidants, including phenolics, ascorbic acid, carotenoids, tocopherols and glucosinolates in lettuce are known to have protective effects against cancer and cardiovascular problems (Nicolle et al., 2004; Liorach et al., 2008). In Thailand, lettuces can be produced all the year round but lettuce production and sales still suffer from quantity and quality problems that are not easily resolved. An important cause of the decrease in the quantity and quality of lettuce is a browning incidence on the leaf surface. This physiological disorder leads to serious economic losses (Saure, 1998). Some researchers have cited that the occurrence of leaf browning is associated with calcium deficiency in the leaves (Saure, 2001) including lettuce (Martyn et al., 2007). This deficiency is associated with the loss of membrane integrity and promotes enzymatic browning (Franck et al., 2007). The damage has long been considered as the main problem in lettuce production because it limits consumer acceptance and decreases the market value (Saltveit, 2000). Therefore, a search for a practical method to decrease this disorder is needed. Saure (2005) reported that calcium is known to stabilize cell membranes. In this way, severity of this physiological disorder would be alleviated. One of the soil amendments that has been shown to decrease this damage is a calcium-releasing compound (Ihl et al., 2003). At present, very little information is available on calcium application in order to decrease the browning damage during lettuce production. Thus, the aim of this study was to investigate the effects of exogenous different calcium formulas at various concentrations applied to Grand Rapids lettuce by soil dressing on controlling the leaf-browning incidence.
MATERIALS AND METHODS
The research was carried out at the experimental field, Division of Agricultural Technology, Faculty of Technology, Mahasarakham University, in the northeast of Thailand in the period from January to March 2009. Seedlings of Grand Rapids lettuce were transplanted at 15 Days after Planting (DAP) and grown singly in 2 L pots filled with a sandy loam soil: rice husk: manure ratio 1:1:1 and placed under field conditions. A Factorial in Completely Randomized Design was arranged and composed of two factors: three calcium types; calcium chloride (CaCl2), calcium nitrate [(Ca (NO3)2)] or calcium oxide (CaO) with five concentrations (0, 0.5, 1.0, 1.5 or 2.0%). Each treatment was carried out in four replicates, ten plants per replication. Calcium was applied by soil dressing to lettuce plants after planting at 20 and 34 days. Untreated plants with calcium served as the control. Basal fertilizer of 15:15:15 at the rate of 20 g per 20 L was watered to the plants every seven days. Plants were sampled at 10 day intervals from 30 to 60 DAP for assessment of five categories: (1) Leaf color was measured with a Hunter Lab Model No. 45/0 L, Serial No. 7092, USA. CIE standard for measuring color values in terms of L* (black = -100 and white = +100), a* (greenness) (- = green and + = red) and b* (yellowness) (- = blue and + = yellow). (2) Phenolic content was performed as described by Ribeiro et al. (2008). Content was expressed as absorption at 765 nm/100 g fresh weight of leaf. (3) Quinone content was extracted as described by Pirie and Mullins (1976). Quinone content was expressed as absorbance at 437 nm. (4) Polyphenol oxidase (PPO) activity determination was carried out according to the method reported by Jiang and Fu (1998). The attained enzyme extracts were measured by spectrophotometer model V-325-XS from China. One unit of PPO activity was defined as the amount of enzyme causing a change of 0.01 in absorbance (420 nm) per 60 sec. (5) Levels of browning on leaf surfaces were scored by determining visually and expressing as a percentage. The collected data were statistically analyzed using the SPSS Computer Programme, Version 6 (SPSS, 1999).
RESULTS AND DISCUSSION
The results were collected after putting calcium with different formulas and various concentrations on lettuce grown under field conditions. The recorded data were composed of:
Leaf color: With respect to leaf color in terms of L*, a* and b* after applying calcium, the results revealed that no significant difference in any of the measured color, in terms of L* values, was observed through 30 to 50 DAP, except for 60 DAP. At the harvesting stage (60 DAP) the results from Table 1 showed a high significant L* values among treatments. Plants treated with calcium, irrespective of any concentrations in forms of CaCl2 and CaO, showed similar L* values. While plants that received Ca (NO3)2 at 1% tended to have the least L* values. For leaf color measured as a* value, the results indicated that most of data showed significant differences through 30 to 60 DAP, except for 50 DAP. At harvest, plants treated with 1.5% CaCl2 had the maximal greenness (a*) of -9.50 (Table 2).
| Table 1: | Color values in term of L* after applying different types and concentrations of calcium |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
While leaf color monitored by b* values showed that there were significant differences in the yellowness (b*) of lettuce’s leaf. At harvest, treatment of 0.5% CaO showed the highest yellowness of leaf color of 33.00 (Table 3).
Phenolic content: The contents of phenolic compound were affected by different calcium applications. The results from Table 4 showed the significant differences in phenolic contents among treatments through plant development. During growth seasons, the content of phenolic compounds in lettuce plants were significantly lower when supplementary 2% CaCl2 was applied. On the harvesting date (60 DAP), plants supplied with 2% CaCl2 exhibited the lowest amount of phenolic compound (0.30 g/100 g FW). While the control plants had a maximum phenolic content of 0.52 g/100 g FW (Table 4).
| Table 2: | Colour values in term of a* after applying different types and concentrations of calcium |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
Quinone content: The results from Table 5 showed that treating with calcium in different forms and concentrations affected quinone contents in leaf extracts of lettuce throughout plant growth. The alteration of quinone substance in lettuce leaves showed the similar trend as phenolic compounds and was significantly affected by calcium treatments. At harvest, the lowest quinone content from plants applied with 2% CaCl2 was observed (Table 5).
PPO activity: The enzymatic browning in lettuce plants were visually detected through all growth periods. The results revealed that activity of PPO from plants treated with different calcium compounds differed significantly among treatments. At 30 DAP, PPO activity decreased markedly and showed the minimum level in plants treated with 2% CaCl2 in comparison to control (Table 6).
| Table 3: | Colour values in term of b* after applying different types and concentrations of calcium |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
When the plants grew up to 40 DAP, the activity of PPO was also the lowest level in plants treated with 2% CaCl2. While the maximum activity of PPO enzymes was found in treatment of 2% Ca(NO3)2 (Table 7). As far as developmental stage at 50 DAP, plants treated with 2% CaCl2 remained the significant lowest activity of PPO (Table 8). At the end of plant growth (60 DAP), PPO activity from plants supplied with 2% CaCl2 sharply decreased to the lowest amount. While the highest level of PPO activity was observed in the plants treated with 2% Ca(NO3)2 on the harvest day (60 DAP) (Table 9). Thus, the results showed similar trends of PPO activities. Throughout PPO activity decreased markedly and showed the minimum level in plants treated with 2% CaCl2 in comparison to control throughout their growth stage. While the greatest activity of PPO was observed from plants treated with 2% Ca(NO3)2, especially at harvesting time (60 DAP).
| Table 4: | Phenolic content of lettuce after applying different types and concentrations of calcium |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05 | |
Levels of browning: Leaf browning was estimated by measuring the extent of the total brown area on leaf surface as a percentage. Degree of leaf browning observed by visual evaluation showed an increasing trend with a plant’s development. Overall browning in lettuce, which marked the visible level of discolouration during plant development, was presented in Table 10. At initial growth stage of 30 DAP, the results showed that browning incidence of 1.33% decreased significantly in plants supplied with 2% CaCl2. The lowest level of browning (5.50%) also found in plants treated with 2% CaCl2 when plants grew up to 40 DAP. In addition, level of browning from plants treated with 2% CaCl2 remained to be the lowest percentage of 5.83% at 50 DAP. At the harvesting stage, the level of browning decreased by 11.17% in plants treated with 2% CaCl2. This implied that there was an apparently beneficial effect of CaCl2 at 2% for controlling these disorders while calcium application in terms of Ca(NO3)2 at 2% had a detrimental effect on browning appearance in lettuce by increasing the browning appearance to the highest degree of 25.33% (Table 10).
| Table 5: | Quinone content of lettuce after applying different types and concentrations of calcium |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05 | |
The effect of different calcium forms with various concentration rates on leaf color, contents of phenolic compounds and quinone, Polyphenol oxidase (PPO) activity and browning percentage was studied in Grand Rapids lettuce. The results provided important data on different responses to different calcium applications, on leaf color during plant growth. For leaf color, there were remarkable differences in leaf color measured as L*, a* and b* after applying different calcium to the soil. At harvest (60 DAP), color measurement, in terms of brightness (L* value), from treatment of Ca(NO3)2 tended to exhibit the lowest values.
| Table 6: | PPO activity of lettuce after applying different types and concentrations of calcium at 30 DAP |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
For greenness, plants treated with 1.5% CaCl2 significantly showed the maximal leaf color in term of a* value while leaf from plants supplied with 0.5% CaO experienced the highest yellowness (b* value). The reason for this may be derived from leaf color being influenced by various calcium supplements. This is probably due to different calcium forms reacting in different ways to leaf color in lettuce. However, CaCl2 at 1.5% was the best treatment on promoting the maximal green color in terms of a* value at harvest. These results were in agreement with Ritchey et al. (1995) who cited that calcium application, in terms of CaCl2, increased the exchangeable ions of Ca2+ inside plant cells leading to activate the metabolism of the chlorophyll molecule in lettuce leaves (Heaton and Marangoni, 1996). While Mitsuya et al. (2000) found that in mesophyll of sweet potato leaves, most thylakoid membranes of chloroplast are lost under salt stress.
| Table 7: | PPO activity of lettuce after applying different types and concentrations of calcium at 40 DAP |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
Further study of the mechanisms of CaCl2 at 1.5% involved in promoting a* value in lettuce may be needed. However, there is very little information available on different calcium formulas concerning leaf color in lettuce production.
For phenolic compound, the results showed that during growth season, the contents of phenolic compounds in lettuce were significantly lower when supplementary 2% CaCl2 was applied. These results showed that calcium applications in term of CaCl2 could reduce phenolic content in lettuce during crop growth. Leaves from plants treated with 2% CaCl2 had the lowest amounts of phenolic content (0.30 g/100 g FW), especially at harvest stage. This is consistent with the report of Lichanporn et al. (2009) who cited that the lower phenolic contents in oriental lily Star Gazer as a result of supplemental different calcium forms with concentration rates.
| Table 8: | PPO activity of lettuce after applying different types and concentrations of calcium at 50 DAP |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
Generally, phenolic compounds in plant tissues are oxidized into quinones under enzymatic catalysis, PPO. Thus, phenolic compounds performed as substrates for PPO activity, which is likely to be involved in the browning reaction (Lichanporn et al., 2009). The destruction of leaf cellular compartmentation allows the phenolic substrates to be accessible to PPO which catalyze the phenolic oxidation and increase susceptibility to browning appearance (Mayer and Harel, 1979). Similarly, Mayer and Harel (1979) reported that the concentration of phenolic compounds is a major factor determining tissue browning development and intensity. If a cell membrane becomes broken, the phenolic substrate will mix with PPO and the browning process will begin. Calcium chloride plays an important role in the stabilization of the cell membrane and maintains the membrane compartmentation, which helps to lower the incidence of browning.
| Table 9: | PPO activity of lettuce after applying different types and concentrations of calcium at 60 DAP |
| Letters within columns indicate least significant differences (LSD) at p* = 0.05, ns: non significant | |
In addition, similar findings of Fletcher et al. (2000) cited that the beneficial effect of CaCl2 on decreasing the susceptibility in plants to stress. These conditions decreased phenolic synthesis (Ruiz et al., 1999). However, the influence of different calcium compounds on the synthesis of phenolic compound in lettuce is still poorly understood. Thus, effect of CaCl2 on reducing the phenolic compound of lettuce warrants further investigation.
With quinone content, interaction between supplementary different calcium formulas with concentration levels was found with respect to quinone content during the growing season. The results from Table 5 showed the alteration of quinone content had a similar trend as phenolic content. Treating with CaCl2 at 2% strongly influenced to decrease both the contents of phenolic compounds and quinone.
| Table 10: | Level of browning occurrence of lettuce after applying different calciums at different concentration |
| Letters within columns indicate least significant differences (LSD) at *p = 0.05, ns: non significant | |
The lowest amount of quinone from plants treated with 2% CaCl2 (0.45 g FW) was observed at harvest. In general, the phenolic compounds could be oxidized to quinone by enzymatic browning, PPO (Degl’Innocenti et al., 2005). As a consequence, these quinones would be condensed and polymerized and responsible for tissue browning (Ke and Saltveit, 1989; Zawistowsky et al., 1991). This experiment confirmed previously published results on positive CaCl2 influence on reducing quinone content of lettuce tissue. Thus, the more content of phenolic compound, the greater quinone formation responsible for the loss of leaf color was observed (Cakmak et al., 1995). These results were not in agreement with Chutichudet et al. (2009) who cited that the contents of phenolic and quinone in lettuce leaves were not affected by putting calcium in terms of gypsum. While pre-harvest calcium supplements with Ca(NO3)2 and CaO have not strongly affected the contents of phenolic compound and quinone. It may be possible that applying the inappropriate calcium compounds promotes the synthesis of these two substances. In addition, application with various calcium substances may affect competitive uptake by root, transport and partitioning within the plant (Grattan and Grieve, 1998). The effects of CaCl2 on the phenolic and quinone depression of lettuce are poorly understood. Thus, the involvement of CaCl2 in the response of plants to lower the phenolic compound and quinone contents needs to be addressed more deeply.
For PPO activity, the results indicated that activity of PPO in lettuce leaf during growing periods was affected by calcium application. From the results of this study, it is clear that the efficiency of calcium on controlling PPO activity in lettuce seems to depend on suitable calcium form and concentration rate. These results implied that each calcium substance applied in this research had a distinct role for controlling PPO activity. Calcium applied as soil dressing in term of CaCl2 at 2% affected to decrease the activity of PPO in Grand Rapids lettuce, throughout the plant growth stage since 30 to 60 DAP (Table 7-10). Generally, PPO are localized in plastids, their phenolic substrates are mainly located in the vacuole so that enzymatic browning only occurs when this sub-cellular compartmentalization is lost (Cantos et al., 2002). Once a deteriorative process (e.g., senescence) is initiated, the compartmentalization of the cell begins to fail (Marangoni et al., 1996). The consequence of this is the mixing of phenol substrates with polyphenol oxidase (Degl’Innocenti et al., 2005). The reduction in PPO activity that was shown from plants supplied with 2% CaCl2 could be due to CaCl2 having a role in metabolic activities, like stabilization of membranes and control of enzyme activity, such as to ameliorate the adverse effects of PPO activity in plants (Arshi et al., 2006). Therefore, it has been suggested that calcium chloride at 2% could protect cell membrane disintegration from the effects of browning reaction. These corresponded with the results of Raese and Drake (2002) who reported that formulas containing calcium chloride were the most suitable for increasing calcium content. In addition, it is also hypothesized that CaCl2 induced ionic balance in plant cells and led to unfavorable conditions to enzyme activity (Beirao-da-Costa et al., 2008). Conversely, a negative effect of Ca(NO3)2 at 2% on promoting the maximum activities of PPO were observed at all growth stages. However, the rise of activity of PPO from plants treated with 2% Ca(NO3)2 may be caused by nitrates toxicity (Shannon et al., 1999). These responsible for accumulation of phenolic compounds leading to an increase in PPO activity which causes an increase in enzymatic browning (Pfeffer et al., 1998). Unfortunately, the underlying biochemistry of enzymatic browning associated with different calcium substances have not yet been fully elaborated. The mechanism by which CaCl2 could interfere with PPO activity is still to be elucidated. Therefore, further study is required to fully understand the effects of CaCl2 in lowering the activity of PPO in lettuce.
For assessing the level of leaf browning as measured by visual evaluation as a percentage, the results from Table 10 reveal that different forms incorporated with concentration rates of calcium supplement had markedly significant effects on leaf browning throughout growth stages, especially at the commercial maturity stage (60 DAP). These results corresponded to the results of Chutichudet et al. (2010) who found that browning appearance in lettuce could be observed visually during the developmental period. Treatment of CaCl2 at 2% had a potential efficiency to reduce leaf browning in lettuce. This trend is supported by Vamos-Vigyazo (1981) and Mayer (1987) who indicated that browning incidence as a consequence of destruction of tissue cellular compartmentation led to a discouragement of PPO activity and to become susceptible to browning disorder. Lettuce is classified as highly susceptible to enzymatic browning (Altunkaya and Gokmen, 2008). In this research, it was found that CaCl2 application at 2% not only decreased the contents of phenolic and quinone but also reduced PPO activity which may primarily be due to the reason that CaCl2 has promontory influence to lower the browning disorder. These corresponded to the results of Fujita et al. (2006) who cited that browning incidence was related to increase PPO activity and levels of phenolic compounds. It is difficult to ascribe a role of different calcium formulas in decreasing browning disorder; however, a role in maintaining cell and membrane integrity has been well established (Poovaiah, 1986). This implies that the role of calcium status in physiological disorder may depend on the cation composition in the substance (Shannon and Grieve, 1999). In addition, it has been hypothesized that development of the browning disorder is a consequence of leaf membrane disintegration (Felicetti and Schrader, 2009). Some nutrients greatly affected the structural and functional integrity of cell membranes. The possible effect of CaCl2 on browning incidence has been reported already in several plants (Carvajal et al., 2000). This could possibly be due to the fact that CaCl2 functioned as stabilizer plant membrane and then delayed the activity of PPO that reduced the browning occurrence. Similar results was found by Manganaris et al. (2005) who reported that CaCl2 could reduce brown rot development in Andross peach fruits. They explained that CaCl2 had a profound effect on delaying the modifications taking place in the cell wall (Brummell et al., 2004). Furthermore, Renault (2005) cited that calcium could alleviate the adverse effect of browning damage in plants by maintaining cell membrane integrity and ion-transport regulation (Picchioni et al., 1995). In contrast, plants receiving 2% Ca(NO3)2 significantly promoted these severe damages throughout the growth periods. These results were in agreement with Grattan and Grieve (1998) who cited that application of some nutrients caused an ion toxicity and ion imbalance that affected plant metabolism and increased the susceptibility to incidences of injury. These results was consistent with Lotze et al. (2008) who found that bitter pit in Golden Delicious apples increased after applying Calcium nitrate [Ca(NO3)2] from early to late applications of fruit growth. Therefore, applications of higher NO3¯ ratios in the nutrient are often associated with increased incidence and severity of browning appearance (Ikeda and Osawa, 1988; Lee et al., 1991). The precise mechanisms of action of these calcium compounds are not yet clear, but passive diffusion across the plasma membrane is involved (Lanciotti et al., 2004). Furthermore, some unknown plant factors might have profound influences on plant ability to absorb and utilize different calcium forms (Fageria et al., 2002). The mechanism of the relationship between CaCl2 and browning incidence in lettuce plants is not clear. More work needs to be done on the mechanisms of CaCl2 on relieving browning incidence and how the CaCl2 at 2% affected to lower this disorder in lettuce.
CONCLUSION
The results showed that 1.5% CaCl2 resulted in significant increase of leaf color in terms of greenness (a* value). A lower significant decrease in the contents of phenolic compound and quinone were noted in plants supplied with 2% CaCl2. In addition, plants treated with 2% CaCl2 also showed the lowest PPO activity and eventually minimum browning incidence at harvesting. These research findings confirmed that soil dressing with calcium in the form of CaCl2 application at 2% is the most effective way to decrease leaf browning in lettuce.
ACKNOWLEDGMENTS
This research was funded by the Mahasarakham University. The authors wish to express their sincere thanks to the Division of Research Facilitation and Dissemination, Mahasarakham University. We gratefully acknowledge Mr. Paul Dulfer for revising the manuscript.