Abstract: A comprehensive study to evaluate calcium, in terms of gypsum (CaSO4.2H2O) by soil dressing application, on enzymatic browning activity of Polyphenol oxidase (PPO) and internal qualities was tested on lettuce var. Grand Rapids under field conditions. A factorial in completely randomized design was arranged with four replications. The results showed that plants-treated with 50 mg kg-1 gypsum applied at 40 DAP had the maximal fresh weight of 25.83 g plant-1. The internal qualities of the lettuce at harvest showed that plants treated with 50 mg kg-1 gypsum had the maximal chlorophyll content (26.80 mg m-2), while all gypsum concentrations applied in this study, had less content of ascorbic acid than the control plants. Plants-treated with 100 mg kg-1 gypsum affected to the lowest level of PPO activity at week 3 after transplanting. Furthermore, gypsum application had no effect to biomass, leaf colour, the contents of phenolic and quinone in lettuce at harvesting stage.
INTRODUCTION
Lettuce (Lactuca sataiva), belonging to the Asteraceae family, is a popular vegetable and considered one of the most important crops all year round in Thailand. In 2007, the total area for growing lettuce in Thailand was 2,119.2 ha with an estimated production of 15,499.87 t ha-1 (Cantos et al., 2002). Most lettuce is used for fresh consumption as fast food and prepared salads. Lettuce contains significant amounts of biologically active components that can impart health benefits beyond basic nutrition. Lettuce is also a major source of dietary antioxidants, including phenolics, ascorbic acid, carotenoids, tocopherols and glucosinolates, which are known to have a protective effect against various forms of cancer and cardiovascular and cerebrovascular diseases (Lister, 2003; Nicolle et al., 2004; Llorach et al., 2008; Verlangieri et al., 1985). Moreover, lettuce is an excellent source of several nutritional properties that are considered as low-acid food or healthier foods (Ahvenainen, 1996; Dupont et al., 2000). Generally, the consumer preference of commercially grown Grand Rapids lettuce is due to its crispiness and attractive bright green colour, but this crop is very vulnerable to enzymatic browning which leads to colour change appearing on the leaf surface; this discoloration has long been considered the main production problem. Colour change may be principally a general indicator of quality because appearance is a major factor that determines lettuce marketability. Altunkaya and Gökmen (2008) cited that lettuce is highly susceptible to enzymatic browning. Thus, leaf browning is a major factor that affects product losses resulting from rejection by consumers, shortening storage life and economic loss (Zhang et al., 2001). Little is known about both the origin of this disorder and its physiological mechanism. Enzymatic browning is mainly associated with polyphenoloxidase (PPO). Oxidation of phenolic compounds to o-quinones is the main cause of browning in fresh lettuces (Macheix et al., 1990; Nicolas et al., 1994). The appearance of this physiological disorders can be observed visually on leaf surfaces (Franck et al., 2007) during the preharvest period (Kays, 1999). However, information on tissue browning susceptibility is limited (Castaner et al., 1999).
Furthermore, enzymatic browning is a direct consequence of membrane disintegration. Therefore, the causes of browning must be sought in processes which affect the membrane integrity (Franck et al., 2007). The advanced maturity stages of plants lead to disruption of the cellular compartments, allowing the substrate and enzymes located in the chloroplast to come into contact (Rocha and Morais, 2001). Enzymatic browning is a direct consequence of membrane disintegration (Kays, 1991; Felicetti and Schrader, 2009). Therefore, the control of browning during plant development, by maintaining the membrane integrity, should be studied to increase the value and quality of harvested lettuce. At present, although much is known about the causes of browning disorder, very little is known about the practical method to control browning disorder in lettuce planted in the field. One of the general soil amendment compounds that has been shown to inhibit browning is calcium-releasing compound included in gypsum or CaSO4.2H2O which has been widely used as a soil amendment in a range of soils. However, there is very little information available on gypsum concerning this disorder on lettuce production. Thus, the purpose of this experiment was to investigate the effect of exogenous soil amendment, gypsum, on browning disorder, PPO activity and other internal qualities of lettuce grown under field conditions.
MATERIALS AND METHODS
The experiment was carried out at the experimental field, Division of Agricultural Technology, Faculty of Technology, Mahasarakham University, in the northeast of Thailand in the period between May to July, 2008. ‘Grand Rapids’ lettuce was planted from seed. The seedlings were transplanted 25 days after planting and grown singly in 2-L pot 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: soil application of slow releasing gypsum (CaSO4.2H2O) at the concentration of 50, 100 or 150 mg kg-1 with different three application times (25, 40 or 55 days after planting, DAP) compared with untreated plants (control). Each treatment was carried out in four replicates, ten plants per replication. Plants were harvested at commercial maturity (65 days after planting) and taken to the laboratory within a few minutes for assessments of: (1) Fresh weight (g), (2) biomass was determined by the method of AOAC (1980) and expressed in percentage. (3) leaf colour was measured on the leaf surface with a Hunter Lab Model No. 45/0-L, Serial No. 7092, USA. CIE standard for measuring colour values L*(black = -100 and white = +100), a* (redness) (- = green and + = yellow) and b* (yellowness) (- = blue and + = yellow) were measured to describe the colour of lettuce’s leaf. (4) The browning appearance on leaf surfaces were scored for evaluating browning as described in González-Aguilar et al. (2004) by determining visually using a scale of 1-5, where 1 = none, 2 = slight, 3 = moderate, 4 = severe and 5 = extreme browning. Degree of browning evaluations was carried out during plant development. (5) Chlorophyll content was determined using a procedure as described by Whitham et al. (1986) and expressed as mg m-2. (6) Phenolic content was performed as described by Ribeiro et al. (2008). The phenolic content was expressed as absorption at 765 nm/100 g fresh weight of leaf. (7) Quinone content was extracted as described by Pirie and Mullins (1976). Quinone content was expressed as absorbance at 437 nm. (8) Total soluble solid content (TSS) (juices being squeezed from flesh tissue using distilled water at ratio 1:3 between flesh and distilled water, measurement was done by digital refractometer (Atago-Palette PR 101, Atago Co., Ltd., Itabashi-ku, Tokyo, Japan). (9) Titratable acidity (TA) evaluation was made by the use of juices described above in number 8 with the method of AOAC (1990). (10) The measurement of pH values was carried out by using juices described earlier in number 8 on pH meter ID 100D, Singapore. (11) Ascorbic acid content was measured by the use of juices described in number 8 with the method of AOAC (1990). (12) 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, China. One unit of PPO activity was defined as the amount of enzyme causing a change of 0.01 in absorbance (420 nm) per 30 sec. The collected data were statistically analyzed using the SPSS Computer Programme, Version 6 (SPSS, 1999).
RESULTS
The results were collected after adding gypsum, with different concentrations of 50, 100 or 150 mg kg-1 at various application times (25, 40 or 55 DAP) for lettuce grown under field conditions. The recorded data were composed of:
Fresh weight: At harvest, there was highly significant fresh weight of product (Table 1). Plant-treated with 50 mg kg-1 gypsum at 40 DAP had significantly the maximal weight of 25.83 g per plant, while the minimum weight was recorded for 150 mg kg-1 at 55 DAP (14.51 g) per plant.
Biomass: The results from Table 1 showed no significant difference in lettuce biomass among treatments with similar mean values of 7.79-14.72%.
Leaf colour: Changes in the leaf colour of the lettuce were monitored by measuring L*, a* and b* at the harvesting stage. From Table 1, there was no significant difference in the leaf colour parameters of L*, a* and b* between the three concentrations of gypsum cooperated with different application times.
Browning appearance: Degree of leaf browning showed an increasing trend with a plant’s developmental time. Leaf browning was observed by visual evaluation on lettuce at the first week after transplanting.
| Table 1: | External qualities of lettuce as influenced by putting gypsum at different concentrations and time application |
| Letters within columns indicate Least Significant Differences (LSD) at **p = 0.01; NS: Non significant | |
At 25 DAP (week 1), the untreated plants showed significantly lower browning level of 0.15. Table 2 also showed the browning appearance of untreated plants was significantly lower than that of the others after transplanting three weeks of 2.56. Afterwards, there was a similar amount of browning in both gypsum treatments and control samples.
Chlorophyll: The results from Table 3 showed a good correlation between gypsum concentrations and chlorophyll contents. On harvesting date (65 DAP), chlorophyll content from plant-treated with 50 mg kg-1 gypsum showed significantly the highest chlorophyll content of 26.80 mg m-2.
Phenolic and quinone content: At harvesting, the total phenolics and quinone contents of the leaf extracts were shown in Table 3. The results revealed that total phenolics and quinone content in lettuce leaves from both plants-treated with different gypsums and control showed the same trend and was not significantly affected by gypsum treatments, ranging from 646.79-1144.11 mg per 100 g and 0.1039-0.2120/mg FW, respectively.
TSS, TA and pH: Gypsum treatment had no significant effect on the internal qualities of the lettuce such as TSS, TA and pH. The mean values of TSS, TA and pH of all treatments at harvesting time ranged from 0.20 -0.28 degree Brix, 0.0320-0.0720% and 6.45-6.55, respectively (Table 4).
Ascorbic acid: Significant differences in ascorbic acid between the plant-treated with gypsum and control were found. All plants-treated with gypsum had the lower ascorbic acid content than control (Table 4).
PPO: The variation in the PPO activity in leaf tissues of lettuce measured at the various developmental stages (week 1, 3 and 5 after transplanting) is shown in Table 5-7. PPO activities in the lettuce leaf were at a very low level at early stage of week 1 after transplanting, then increased obviously in week 3 and finally decreased slightly at week 5. Significant differences (p>0.05) of PPO activities were not found between treatments on week 1 and week 5, except for three weeks after transplanting, PPO activity tended to increase progressively and statistical differences among the gypsum concentrations were observed. From Table 6, PPO from lettuce-treated with 100 mg kg-1 gypsum showed the lowest activity.
| Table 2: | Browning scores of lettuce as influenced by putting gypsum at different concentrations and time application |
| Letters within columns indicate Least Significant Differences (LSD) at *p = 0.05; NS: Non significant | |
| Table 3: | The content of chlorophyll, phenolic and quinone of lettuce as influenced by putting gypsum at different concentrations and time application |
| Letters within columns indicate Least Significant Differences (LSD) at *p = 0.05; NS: Non significant | |
| Table 4: | TSS, TA, pH and ascorbic acid content of lettuce as influenced by putting gypsum at different concentrations and time application |
| Letters within columns indicate Least Significant Differences (LSD) at *p = 0.05; NS: Non significant | |
| Table 5: | Activity of PPO lettuce as influenced by putting gypsum at different concentrations and period of time one week after transplanting |
| NS: Non significant | |
| Table 6: | PPO activity of lettuce as influenced by putting gypsum at different concentrations and period of time three weeks after transplanting |
| Letters within columns indicate Least Significant Differences (LSD) at *p = 0.05; NS: Non significant | |
| Table 7: | PPO activity of lettuce as influenced by putting gypsum at different concentrations and period of time five weeks after transplanting |
| NS: Non significant | |
DISCUSSION
The effect of pre-harvest soil application of gypsum on the development of enzymatic browning and internal qualities of Grand Rapids lettuce was studied. Fresh weight of plants treated with 50 mg kg-1 gypsum applied at 40 DAP had the maximal plant weight, but the gypsum had no effect to biomass. This implied that fertilization with gypsum improved only fresh weight. This may be attributed to calcium that can increase the cell wall and the integrity of the plant membrane, regulate ion transport and control ion exchange particularly in meristematic tissue (Rengal, 1992; Gislerod, 1999). Ritchey and Snuffer (2002) reported that gypsum is a readily available calcium amendment that is sufficiently soluble to move rapidly into the soil when surface-applied. The solubility of gypsum, when surface applied, permits a quick release of calcium (Ca2+) and sulfate (SO42-) ions into the soil solution (Dontsova et al., 2005). Thus, use of gypsum will have a positive effect on lettuce growth. These results were consistent with those reported in blueberry (Wright et al., 1993), citrus (Bañuls et al., 1991) and maize (Cramer et al., 1988). The EPA (2008) reported that gypsum has been recognized as a nutrient source of sulfur, which is essential for many crops. Gypsum also improved soil physical properties and allowed for greater water infiltration in soils (Dontsova et al., 2005; EPA, 2008). Some additional benefits of gypsum application may result in increases in the exchangeable Ca/Al ratio (Ritchey and Snuffer, 2002). However, a more likely alternative explanation for yield improvement may be attributed to gypsum enhanced mineral nutrient uptake due to reduced Al phytotoxicity (Stout and Priddy, 1996). However, the appropriate time for gypsum application in this experiment should be taken at 40 DAP because gypsum is considered to be long-lasting effects, which may be directly reflected on fresh weight (Prado et al., 2005).
For leaf colour, the results showed that at the harvesting stage, there was not a remarkable difference in leaf colour in terms of L*, a* and b*, while chlorophyll content in the lettuce leaves were increased significantly by application of gypsum treatment to the soil. The reason for this may be derived from exchangeable ions of Ca2+ and Mg2+ levels increased with the gypsum treatments (Ritchey et al., 1995) leading to activate the metabolism of the chlorophyll molecule in lettuce leaves (Heaton and Marangoni, 1996; Heaton et al., 1996). Prado et al. (2005) reported that liming treatments were most effective in delaying the degradation of the green pigments (chlorophyll) of guava fruit by acting to retard senescent changes.
The results on browning appearance showed that lettuce leaves had the higher scores of browning appearance. Similar results were found by Soares et al. (2005) who verified that maturity stages had a strong influence on the incidence of internal browning of pineapple fruit. For gypsum concentration, untreating with gypsum showed a marked difference in leaf browning scores by visual evaluation in only the early stage after transplanting. The opposite result was found by Dilley (1990) and Hewett and Watkins (1991). They reported that calcium application could reduce the incidence of this disorder in apples. It is possible that another variable conditions in the field, especially environmental conditions, could have effect to these variable responses.
For the results on the content of phenolic, quinone, TSS, TA and pH, it was found that all of these parameters in the lettuce leaves were not affected by gypsum application. Unfortunately, there has been a lack of recorded data about these parameters. This may be due to internal qualities of lettuce being influenced by numerous factors, such as environmental factors (Cheynier et al., 1998; Liu et al., 2007) and degree of maturity (Montealegre et al., 2006; Manach et al., 2004). These results were similar with the previous researches of Kahkonen et al. (1999) who found no relationship between antioxidant activity and phenolic content in some plant extracts. Other authors have reported that browning is not necessarily related to phenolic content in fruit species such as apples (Couture et al., 1993; Weurman and Swain, 1955). For quinone content, Aude et al. (1990) cited that the o-quinone was quite unstable and highly reactive electrophilic molecules that spontaneously polymerize, leading to the formation of brown pigments responsible for tissue browning (Ke and Saltveit, 1986, 1989; Gawlik-Dziki et al., 2008). While Degl’Innocenti et al. (2007) found that during the developmental stage, phenol and quinone content did not vary, possibly as a result of continuous conversion between these molecules through an enzymatic spontaneous reaction. Furthermore, no significant difference in the content of TSS, TA and pH were observed in the leaves of lettuce at harvesting stage. These results were in agreement with the results reported by Prado et al. (2005) whom reported that liming did not significantly affect on some chemical characteristics of guava fruit. Unfortunately, little information of the internal qualities involving browning appearance present in lettuce has been available during plant development.
The results on ascorbic acid content in plant-treated with gypsum showed lower than control plant. This implied that there was an apparently greater detrimental effect of gypsum on the ascorbic acid content. The results raised a question on the reason for the differences in ascorbic acid contents between treatments. Unfortunately, little information of the ascorbic acid presented in lettuce has been available during plant development. These are corresponded to the results of de Castro et al. (2008) cited that ascorbic acid in plant tissues and calcium may worked together preserving the stability of the membrane and simultaneously decreased ascorbic acid content in plant-treated with gypsum. Thus, it is doubtful that gypsum is associated with still unknown factors and might be an indirect effect to ascorbic acid degradation. Generally, ascorbic acid acts as an antioxidant as an enzyme inhibitor because it reduces the initial quinone formed by the enzyme to the original diphenol before it undergoes secondary reactions, which lead to browning (Rapeanu et al., 2006). The content of ascorbic acid in vegetables can be influenced by various factors including preharvest climatic conditions, cultural practices and maturity stage (Lee and Kader, 2000). Our results were consistent with the reports of Lee and Kader (2000) who reported that ascorbic acid was also easily oxidized, especially greatly favored in aqueous solutions of alkaline pH, which is in contrast to Macnish et al. (2000), who found that the applied calcium exerts its stabilizing function (Tobias et al., 1993), affecting to reduce the rate of ascorbic acid degradation (Conway and Sams, 1983). However, the exact mechanism of gypsum on decreasing the ascorbic acid content is not yet clear. Further work is required to better understand the biochemical mechanism of gypsum-induced ascorbic acid loss in Grand Rapids lettuces because most successful vegetable growers are careful to properly use the fertilizer to obtain good quality and maintain their nutritional value. The results on PPO activities of lettuce plants were quite similar during development and relatively unaffected by treatment with gypsum, except at the third week after transplanting. At this period, activities of PPO in leaves of the control plants were higher than in the plants fertilized with 100 mg kg-1 gypsum. This could possibly be due to a role for calcium in gypsum maintaining the stability of membranes in plant cell, thus delaying senescence and controlling physiological disorders in lettuces (Poovaiah, 1986). These corresponded to the results of Zheng et al. (2006) who cited that calcium content in apples was characterized by dynamic changes. They showed that the calcium concentration in apple leaves decreased with the developmental stage, thus the control of enzymatic browning was decreased (Poovaiah, 1986) because enzymatic browning is a direct consequence of membrane disintegration (Franck et al., 2007). The destruction of cellular compartmentation allows the phenolic substrates to be accessible to PPO which catalyze the phenolic oxidation and form the browning disorder (Mayer, 1987). Thus, the application of gypsum, which is composed of calcium, probably acted to retard senescent changes by preserving membrane integrity (Picchioni et al., 1996) and thereby decreased the enzymatic browning by PPO activity (Xuan et al., 2001). Similar results concerning change in PPO activity was reported for Feizixiao fruit (Hu et al., 2000). This could be explained by, with the extension of development, cells gradually lose the integrity of the membrane system, which results in the loss of compartmentation, allowing PPO enzymes, which solubilized from the plastid to the cytoplasm, act with phenolic compounds leaked from the vacuole leading to quinone formation and polymerization of brown pigments (Zhang et al., 2000; De Castro et al., 2008). In addition, calcium uptake by root was rapid and linear in the early developmental stage for cell wall stabilization and membrane integrity, but then declined distinctly, continuing until harvest (Cline et al., 1991). Thus the later the plant development, the more degree of browning observed. Furthermore, calcium applied to the potting medium could increase calcium levels in tissue susceptible to enzymatic browning during rapid development (Harbaugh and Woltz, 1989). At present, the underlying biochemical factors associated with an enzymatic browning disorder of lettuce focused on gypsum application to modulate PPO enzyme activities are poorly understood. Moreover, there is no report about the efficiency of gypsum for controlling the enzymatic browning in lettuce during development. Thus, it is difficult to ascribe the role of gypsum in modulating PPO activity; however, a role in maintaining cell and membrane integrity has been well established (Poovaiah, 1986). Thus the major role of gypsum affected to PPO in catalyzing the enzymatic browning of lettuce requires further investigation. In any further investigation, the application time may be more suitable if it reduced ten days (i.e., 25, 35 and 45 DAP). Other factors involving browning disorder of lettuce should be examined.
In conclusion, it was found that soil gypsum at 50 mg kg-1 applied at 40 DAP had the maximal fresh weight. At harvesting, lettuce-treated with 50 mg kg-1 had the highest chlorophyll content. In addition, plants treated with gypsum at different concentrations had lower ascorbic acid content than the control, while 100 mg kg-1 gypsum lowered the PPO activity at week 3 after transplanting. Furthermore, treating with gypsum had no effect to the following parameters of biomass, leaf colour, the content of phenolic, quinone, TSS, TA and pH in lettuce.
ACKNOWLEDGMENTS
This research was funded by the Mahasarakham University under project no. 5101074/2551. The authors wish to express their sincere thanks to the Financial Office for financial assistance. We gratefully acknowledge Mr. Paul Dulfer for revising the manuscript.