Impaginato 49 Adv. Hort. Sci., 2018 32(1): 49-59 DOI: 10.13128/ahs-21360 Partial root-zone irrigation effects on growth, metabolism and calcium status of Mangosteen seedling (Garcinia mangostana L.) D.P. Hapsari, R. Poerwanto(*), D. Sopandie, E. Santosa Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University, Bogor, Indonesia. Key words: drought, low soil moisture, photosynthetic rate, tropical plant, water manage- ment. Abstract: Efficient irrigation technique for mangosteen seedling was evaluated, from October 2016 to May 2017, in order to determine the growth and mor- phophysiology of both the conventional deficit irrigation (CD) and partial root- zone irrigation (PR). A set of randomized block design, with 4 replicates each, was applied on 100% field capacity (control), 50% field capacity (CD1), 30% field capacity (CD2), and ratios of 100:50% field capacity (PR1), 100%:30% field capacity (PR2) and 50:30% field capacity (PR3). The results showed a restriction in mangosteen growth, except control, as indicated by decrease in total dry mass, which due to decrement in leaf number, photosynthetic rate and root growth. Malondialdehyde (MDA) level and glutathione peroxidase (GPX) activi- ty was higher while proline accumulation was lower in PR compared to those of both CD and control treatments. Secondary metabolites content changes in treatments, such as octacosane, cysteamine sulfonic acid, propyl oleate, 1-nan- odecene, and 2-butyn-1-ol-4metoxy were synthesized in the low soil moisture conditions. Leaf Ca-pectate, Ca-phosphate and dissolved Ca tended to increase in low soil moisture. The PR1 treated plant had the highest water use efficien- cy. Therefore, PR technique could be applied when the soil moisture level rep- resents 50% (or more) of the field capacity. 1. Introduction Mangosteen (Garcinia mangostana L.) is a tropical perennial crop that plays an ecologically important role in agroforestry system (Wijayanto and Hartoyo, 2015). It produces an exotic fruit with high antioxidant level (Kurniawati et al., 2010). However, many producing countries, such Indonesia, Malaysia, Thailand and India (Osman and Milan, 2006), are fac- ing both fluctuations in production and yellow latex matter in mangos- teen fruits production (Sdoodee and Limpun-Udom, 2002; Poerwanto et al., 2010). Matra et al. (2016) reported that mangosteen exhibits moder- ate genetic variation within a population. On the other hand, according to (*) Corresponding author: roedhy8@yahoo.co.id Citation: HAPSARI D.P., POERWANTO R., SOPANDIE D., SANTOSA E., 2018 - Partial root-zone irrigation effects on growth, metabolism and calcium status of Mangosteeen seedling (Garcinia mangostana L.). - Adv. Hort. Sci., 32(1): 49-59 Copyright: © 2018 Hapsari D.P., Poerwanto R., Sopandie D., Santosa E. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 18 October 2017 Accepted for publication 13 December 2017 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(1): 49-59 50 Martias and Mansyah (2014), the variations in man- gosteen quality were affected by seasonal variation, water availability, and cultivation techniques. Furthermore, Poerwanto et al. (2010) stated that high water fluctuation in the soil will affect the turgor pressure so that the duct secretory of yellow latex will break out and contaminate the fruit. As other tropical fruits, mangosteen requires low soil moisture root zone to promote flowering (Paull and Nakasone, 1998). However, extended low water status might adversely affect the plant growth (Mustaha, 2012). Preventing water fluctuation on mangosteen root zone is not easily carried out due to the fact that mangosteen is usually planted in an arid area (and depend on rainfall). On the other hand, mangosteen was spread in hills area with agro- forestry system, making the irrigation setting diffi- cult. Thus, the management of irrigation becomes an important factor. However, mangosteen is allegedly non-responsive to irrigation due to the uniqueness of its root mor- phology (Wiebel et al., 1994). Indeed, irrigation was observed to be ineffective in decreasing the water fluctuation (Sdoodee and Chiarawipa, 2005). Besides, the root growth of mangosteen is both slow and sea- sonal and it grows faster before the appearance of new leaves, steadily decreases during the leaves development, and stops post-dormancy period (Hidayat, 2005). Thus, an efficient irrigation tech- nique is needed to decrease water fluctuation. Partial root-zone irrigation (PRI), often referred to as partial root-zone drying (PRD), is a well-known irrigation method which alternately irrigates the root zone (Sepaskhah and Ahmadi, 2010). Adwirman (2006) has already applied the PRD technique on mangosteen but further studies are still needed on both physio- logical responses and nutrient status of the plant. In present study, the calcium status was also analyzed, either in the form of dissolved, pectate, phosphate and oxalate. In fact, calcium is one of important mineral in mangosteen, especially in relation to yellow latex (Dorly et al., 2011; Kurniadinata, 2015) and plays an important role in the mechanism of adaptation to stress condition (Liu et al., 1998; Chen et al., 2002), especially on signal transduction in the responses to water deficit (Hong-Bo et al., 2008). Its status was analyzed in the present research to determine the correlation between the plant root water status and the occurrence of yellow latex, especially Ca-complex such as Ca-oxalate (Korth et al., 2006; Setyaningrum, 2011), Ca-dissolved, phosphate, and pectate (Saure, 2005). Therefore, this study aims to: (1) determine the growth and morpho-physiological responses of mangosteen seedlings, (2) determine the role of cal- cium, and (3) evaluate the level of secondary metabolites in different water status conditions. 2. Materials and Methods Orchard and plants Two-years old mangosteen seedlings at averages height of 30±2.02 cm and leaves number, ranged from 17 to 20, were planted on Pasir Kuda experi- mental field (±260 m asl), Bogor, Indonesia between October 2016 and May 2017. A set of randomized block design, with 4 replicates, was used for field capacities of both conventional deficit irrigation (CD) and partial root-zone irrigation (PR) methods, com- prised of 100% (control), 50% (CD1), 30% (CD2), 100% A: 50% B (PR1), 100% A: 30% B (PR2) and 50% A: 30% B (PR3). Mangosteen seedlings were planted in root-boxes (50 x 40 x 20 cm) in accordance with each treatment. One side of the root-box was made of glass and covered with a black thick cloth to observe the root growth, in a non-destructive way. The root-box was divided into two parts (Fig. 1) in the PR treatment and the partitions were layered by hydrophobic plastic material, in order to avoid water flow from one side to the other side. Both sides of the root-box were filled with soil and compost at a ratio of 1:1 (w/w). Mangosteen roots were carefully cleaned and divided into two symmetrical parts and planted on both side A and side B of the root-box, respectively. The planted mangosteen seedlings were acclimatized and watered within the field capacity conditions for four weeks. In the last week of the acclimatization period 100 g dolomite [CaMg(CO3)2; ±30% CaO] was applied in each root-box. Fig. 1 - Root-box illustration in conventional deficit (A) and par- tial root-zone (B) irrigation treatment. In the PR treat- ment, the root was planted on 2 sides, side A and side B. Hapsari et al. - Partial root-zone irrigation 51 Field capacity determination Soil water content (WC) and humidity (RH) were measured to quickly determine the field capacity (FC). Soil water content was determined by gravimet- ric method (Abdurachman et al., 2006). A hundred grams of soil was weighed (FW) and heated at 105oC for 24 hours (DW) and the soil water content calcu- lated with the following equation: FC = (FW - DW) / DW x 100% Soil RH was measured by means of a soil moisture meter (HT5213, China). Soil water content and RH were measured every 3 days for the optimization (Table 1). Water treatment was applied based on Table 1. The 100% field capacity occurred when the condition was 64.39% for the soil water content and 10 for the RH. The 50% and 30% field capacity treat- ments were obtained at a soil water content of 28.16% (day 13) and 20.06% (day 25), respectively. In order to simplify the water availability application, the watering syllabus was set for every 2 days, 2 weeks and 3 weeks for the 100%, 50% and 30% field capacity treatments. Soil moisture meter was installed on root-box to control the soil condition. Measurements Leaf water potential and relative water content. Healthy mature leave samples were taken at 7.00 A.M., placed into sealed plastic, and kept in a cooler box for further observation in laboratories. The leaves were cut in a cup and measured in a WP4 chamber and leaf water potential was measured using a WP4 Dewpoint Potential Meter (Decagon Devices Inc, USA). The relative water content (RWC) measurement of leaves was carried out in accor- dance with the leaf water potential. The samples used in the leaf water potential measurement (the fresh weight/FW measured) were soaked in the cup containing distilled water. The surface of the cup was covered with filtering paper so that the leaves do not float. Afterwards, the cup was kept in a cool storage for 24 hours, then drained and weighed to determine the turgid weight (TW). Leaves samples were dried at 70°C for 3 days and weighed to determine the dry weight (DW). The RWC was calculated by means of the following equation: RWC = [(FW-DW) / (TW-DW)] x 100 Photosynthesis and transpiration rates . Photosynthesis and transpiration rate were mea- sured prior to treatment and 2 months post-treat- ment using LI-COR 6400 (LI-COR Inc, USA). Calcium content. Calcium content in dissolved form was determined following the method devel- oped by Suwwan and Poovaiah (1978), while calcium in complexed forms was measured gradually accord- ing to Chen and Uetomo (1976) procedure. Malondialdehyde (MDA) level. Lipid peroxidation activity was determined from MDA content that was measured by mean of Wang et al. (2013) procedure. Briefly, 0.4 g leaves sample was homogenized with 10 ml TCA. Homogenate was centrifuged at 4oC for 10 min at 3000 g. Then, 2.5 ml supernatant was added to the reagent which is containing of 0.5% TBA and 20% TCA to be incubated at 80°C for 25 min. The absorbance was read at 440, 532 and 600 nm. MDA level was calculated with the following equation: MDA = 6.45 (A532-A600) - (0.56 x A440) Proline content. Proline content was measured following the method developed by Bates (1973). In brief, 0.5 g leaves sample was homogenized with 3% sulfosalicylic acid. The homogenate was centrifuged at 12.000 g for 10 min. The supernatant was mixed with reagent which contains ninhydrin and glacial acetic acid. The mixture was incubated at 100°C for 60 min and transferred into an ice bath immediately. Afterwards, sample was extracted with 4 ml toluene and stirred in vortex. The absorbance was read at 520 nm. Proline concentration was calculated using proline standard curve. Glutathione peroxidase (GPX) activity. GPX activity was analyzed following the method developed by Urbanek et al. (1991). In brief, leaf were extracted in phosphate buffer. The reaction mixture containing Table 1 - Optimization of soil water content and humidity of mangosteen root zone during drought stress to determine the watering syl- labus Numbers, within the same row, followed by the same letters showed no significant differences based on DMRT at a probability level of 5%. Bold numbers indicating 100%, 50% and 30% field capacity, consecutively. Variables Period of drought stress (days) 1 4 7 10 13 16 19 22 25 Water content (%) 64.39 a 43.06 b 44.24b 41.37 b 28.16 c 27.43 c 24.19 cd 21.12 d 20.06 d Relative humidity (1-10) 10.00 a 8.68 b 8.41 bc 8.04 bc 7.60 c 7.60 c 5.71 d 5.71 d 5.53 d Adv. Hort. Sci., 2018 32(1): 49-59 52 phosphate buffer (pH 7.0), EDTA, guaiacol, H2O2 and 50 l enzyme extract. The enzymatic reaction was initi- ated by addition of extract and the increase in absorbance recorded at 470 nm for 1 min. Enzyme activity was quantified by the amount of tetraguaia- col formed using its molar extinction coefficient (26.6 mM-1 cm-1). Secondary metabolites content. Secondary metabolites was analyzed in Jakarta Regional Health Laboratory using GCMS on fresh mature leaves from control and the most severe (PR3) treatment. Plant growth and biomass. The length and volume of the root, the fresh and dry weight of plant were observed 2 months post treatments. Plants were cleaned prior to observation. The root variable obser- vation, in PR treatment, was carried out by merging both side A and side B. The length of root was mea- sured from the boundary between root and main stem. The volume of root was measured based on Archimedes principle. Plant was weighed to measure the fresh weight then heated at 80oC for 72 h to determine the dry weight. Statistical analysis Data were analyzed with F test and Duncan Multiple Range Test (DMRT) at a probability level of 5% using Statistical Analysis System 9.4 (SAS 9.4M4) software. 3. Results The leaves in all treatments, except control, with- ered on three weeks post-treatment (Fig. 2). However, the plants became fresh again after re- watered, except in CD2 and PR3 treatments. Besides, abortion and drying leaves were also observed in mangosteen seedlings. Abortion of old leaves was more severe in PR2 and PR3 treatments. In fact, stressed mangosteen leaf has a unique dried pattern. The whole leaves of the stressed mangosteen seedling did not dry entirely, indeed, it dried step by step starting from both the edge and tip of the leaf blade (Fig. 2G). The reduction in leaves number was observed since the first month of the treatment; the decrease was more severe during the second month, especial- ly in CD2, PR2, and PR3 treatments (average 9.1, 8.7, 7.2 leaves per plant, respectively) compared to con- trol which had 14.9 leaves (Table 2). PR1 treatment did not indicate any severe leaves abortion. Thus, there were no significant differences in terms of leaves number between control and PR1 treatments from the beginning until the end of the treatment period (Fig. 2 and Table 2). Fig. 2 - Mangosteen seedling canopy in different water availabi- lity treatments (2 months post treatment). A= control, B= conventional deficit irrigation, 50%, C =conventional defi- cit irrigation, 30%, D= partial root-zone irrigation 100% side A: 50% side B, E= partial root-zone irrigation 100% side A: 30% side B, F= partial root-zone irrigation 50% side A: 30% side B, G= dried pattern on mangosteen leaf). Field capacity Number of leaves 0 MAT 1 MAT 2 MAT Control (100%) 18.2 16.6 a 14.9 a CD1 (50%) 18.2 15.2 abc 10.8 b CD2 (30%) 19.2 14.7 bc 9.1 c PR1 (100%A:50%B) 19 16.0 ab 13.6 a PR2 (100%A:30%B) 18.3 14.0 c 8.7 c PR3 (50%A:30%B) 18.4 14.6 bc 7.2 d F-test NS * * Table 2 - Decreasing in mangosteen leaves number in different water availability treatments Numbers, in the same column, followed by the same letter indi- cates no significant differences based on DMRT at a probability level of 5%. MAT= month after treatment. Mangosteen leaf water potential ranged from -2.95 to -3.59 MPa before the treatments (Table 3). In present research, the leaf water potential above -3.59 MPa in two months after treatment was classi- fied as a stress condition for seedlings, coincide with the leaf morphological characteristics which showed withered condition (Fig. 2). Leaf water content decreased post-drought treatment in both CD and PR conditions (Table 3). The lowest water potential was observed in PR3 treatment (-4.72 MPa) which had a field capacity of 50% on side A and 30% on side B. On the other hand, leaf water content also decreased in Hapsari et al. - Partial root-zone irrigation 53 all treatment compared to control, although the decreasing was not significant (α 5%), except for PR3 treatment which had the lowest leaf water content (Table 3). Drought treatment decreased both photosynthe- sis and transpiration rates of mangosteen seedlings two months after treatment (Table 4). PR treatments showed the lowest photosynthesis and transpiration rate and, among PR treatments, PR3 recorded the worst performances in terms of photosynthesis and transpiration rate (6.06 µmol CO2 m-2 s-1 and 0.02 Mmol H2O m-2 s-1, respectively). The dry weights of canopies and roots were signif- icantly lower in CD and PR treated plants than control (Table 5). Unexpectedly, PR1 treatment tended to have the lowest decrement in dry weights of the canopy, roots and total plant. On the other hand, the highest decrements were observed in CD2 and PR2 treatments on both canopy and total plant dry weight, although statistically similiar. Control and PR1 treatments had the longest root apparatus (Fig. 3), although they did not differ significantly (Table 6). Drought stress significantly restricted the root growth in CD1, CD2 and PR3, excepted PR1 and PR2 treatments. Meanwhile the root volume did not show any differences among treatments. MDA content in CD1, CD2 and PR1 treatments did not differ significantly to that of the control treat- ment (Fig. 4). PR2 and PR3 treatments had the high- Table 3 - Water potential and content of mangosteen seedling leaf in different water availability treatments at the beginning (0 months) and 2 months post-treatment Numbers, in the same column, followed by the same letter indi- cates no significant differences based on DMRT at a probability level of 5%. MAT= month after treatment. Treatment (FC) Leaf water potential (Mpa) Leaf water content (%) 0 MAT 2 MAT 0 MAT 2 MAT Control (100%) -2.95 -3.09 a 81.71 97.57 a CD1 (50%) -2.96 -4.00 b 77.5 71.62 ab CD2 (30%) -3.00 -3.99 b 73.93 53.55 ab PR1 (100%A:50%B) -3.59 -4.12 b 69.51 56.89 ab PR2 (100%A:30%B) -3.37 -4.51 b 68.08 58.81 ab PR3 (50%A:30%B) -3.53 -4.72 b 68.04 31.68 b F-test NS * NS * Table 4 - Photosynthesis and transpiration rates of mangosteen seedling in different water availability treatments at the beginning (0 months) and 2 months post-treatment Numbers, in the same column, followed by the same letter indi- cates no significant differences based on DMRT at a probability level of 5%. MAT= month after treatment. Treatment Photosynthesis rate (µmol CO 2 m-2 s-1) Transpiration rate (Mmol H 2 O m-2 s-1) 0 MAT 2 MAT 0 MAT 2 MAT Control (100%) 16.23 16.20 a 0.52 0.21 a CD1 (50%) 15.49 11.76 ab 0.49 0.11 ab CD2 (30%) 18.27 12.05 b 0.56 0.05 ab PR1 (100%A:50%B) 15.51 8.35 cd 0.62 0.07 ab PR2 (100%A:30%B) 15.56 10.42 bc 0.55 0.05 ab PR3 (50%A:30%B) 15.33 6.06 d 0.61 0.02 b F-test NS * NS * Table 5 - Dry weight of mangosteen seedling canopy, root and total plant in different water availability treatments at 2 months post- treatment Numbers, in the same column, followed by the same letter indicates no significant differences based on DMRT at a probability level of 5%. Treatment Dry weight (g) Relative decrease to control (%) Canopy Root Total Canopy dry weight Root dry weight Total dry weight Control (100%) 12.84 a 5.15 a 16.43 a - - - CD1 (50%) 9.20 b 3.08 b 12.28 b 28.35± 0.15 40.19±1.02 39.80±0.11 CD2 (30%) 7.44 b 3.33 b 10.77 b 42.06±0.11 35.34±1.00 47.21±0.10 PR1 (100%A:50%B) 9.65 b 4.83 ab 13.98 ab 24.84±0.11 6.21±0.73 31.47±0.13 PR2 (100%A:30%B) 6.99 b 3.87 ab 10.49 b 45.56±0.07 24.85±1.14 48.58±0.01 PR3 (50%A:30%B) 8.30 b 3.49 b 12.17 b 35.36±0.08 32.23±2.05 40.34±0.03 F-test * * * - - - Fig. 3 - The root system of mangosteen seedling in different water availability treatments (2 months post treatment). C= control), CD1= conventional deficit irrigation 50%, CD2= conventional deficit irrigation 30%, PR1= partial root-zone irrigation 100% side A:50% side B, PR2= partial root-zone irrigation 100% side A:30% side B, PR3= partial root-zone irrigation 50% side A:30% side B. Adv. Hort. Sci., 2018 32(1): 49-59 54 est MDA content, being 1.033 and 1.501 µmol/ml respectively, which were significantly different to that of the control treatment. Proline accumulation was in accordance with that of MDA content with the highest value in PR3 treatment followed by CD1 and CD2 (Fig. 4). Proline content in PR1 and PR2 treat- ments did not show any significant different to that of the control treatment. Glutathione peroxidase (GPX) activity showed different result compared to proline and MDA. In fact, the control treatment had the highest GPX activity showed not significantly dif- ferences among treatments, except PR3 treatment (Fig. 4), as a result of severe stress. Mangosteen seedling, on limited water condition, produced more diverse secondary metabolites than the control, especially terpenoid and fatty acid (Table 7). Five secondary metabolites were found in the leaves of control plant, i.e. oleic and hexadecenoic acid (from the fatty acid group), squalene, vitamin E and neophytadiene (from terpenoid group). Vitamin E and squalene content increased in stressed man- gosteen seedling by 28% and 62% respectively, while oleic acid content decreased by 15%. On the other hand, hexadecenoic acid and neophytadiene were not detected in stress plant. There were 2 kinds of unknown compounds produced in severe stress, i.e. 1-nonadecene and 2-butyn-1-ol 4 metoxy. Dissolved Ca was mostly found in mature leaves, young leaves, roots, and branch, while Ca-pectate and Ca-phos- phate were mostly found in mature leaves, roots, young leaves and branch (Table 8). Table 8 shows that both dissolved and pectate calcium contents were high in severely stressed plants, especially in young and mature leaves. Furthermore, the leaves calcium content in dissolved and pectate form of CD1 treatment were significantly higher than that of con- trol. In CD2 treatment, calcium, in dissolved and pec- tate form, was tended to be lower compared to CD1 treatment but not significantly different from the control. A similar result was observed in dissolved calcium content of mangosteen seedling roots, while no differences in calcium content in branch, was observed. Table 8 also shows that Ca-phosphate and Ca-oxalate in branch were significantly higher in all PR treatments compared to control, while there were no differences in Ca-oxalate content in leaves and roots. Table 6 - Root length and volume of mangosteen seedling in dif- ferent water availability treatments at 2 months post- treatment Numbers, in the same column, followed by the same letter indi- cates no significant differences based on DMRT at probability level of 5%. Treatment Root length (cm) Root volume (ml) Control (100%) 35.00 ab 11.67 CD1 (50%) 32.70 bc 8.33 CD2 (30%) 29.45 cd 6.33 PR1 (100%A:50%B) 37.75 ab 10.33 PR2 (100%A:30%B) 33.30 abc 7.67 PR3 (50%A:30%B) 28.00 d 7.5 F-test * NS Fig. 4 - MDA (A), proline (B) content and GPX activity (C) of man- gosteen seedling in different water availability treat- ments (2 months post-treatment). Numbers followed by the same letter indicates no significant differences based on DMRT at a probability level of 5%. Table 7 - Secondary metabolites content in control and PR3 treatments of mangosteen seedling 2 months post- treatment Secondary metabolite compound Group of compounds The existence Control PR3 Oleic acid Unsaturated fatty acid ++ ++ Iliadic acid Unsaturated fatty acid - + Hexadecenoic acid Saturated fatty acid + - Squalene Terpenoid + + Vitamin E Terpenoid + + Neophytadiene Terpenoid + - Octacaine Acyclic hydrocarbon - + Cysteaminesulfonic acid Amino acid - + Propyl oleate Ester fatty acid - + 1-nonadecene Unknown - + 2-butyn-1-ol, 4 methoxy Unknown - + - not detected, + peak area below 20%, ++ peak area between 20-50%, PR3= partial root-zone irrigation 50% side A:30% side B. Hapsari et al. - Partial root-zone irrigation 55 4. Discussion and Conclusions In the present study, mangosteen leaves changes its morphology and aborts as a consequence of drought stress in different water treatments. Leaves abortion was more marked in CD2, PR2 and PR3 treatments, a common symptom of plants under drought stress (Munne-Bosch and Alegre, 2004). It was expected that the leaves of CD2, PR2 and PR3 treatments would accumulate more ABA and trigger the abscission process (Wingler and Roitsch, 2008; Peleg and Blumewald, 2011). On the other hand, leaves abortion in control and PR1 treatments were not significantly different from the beginning until the end of the treatment period, indicating that PR1 were not severely water stressed. Morphological changes and leaves abortion suggest that mangos- teen seedlings were less tolerant to drought stress. However, mangosteen showed a low response to drought stress since the withered leaves occurred on 3 weeks post-treatment. The low response in man- gosteen was expected to be a consequence of man- gosteen seedling low growth as stated by Ramlan et al. (1992). An high relation between leaf morphology and changes in water potential due to the variations in terms of water treatments was observed. It is expect- ed to be the mechanism of mangosteen adjustment by decreasing water potential in tissues in order to absorb the water in the soil. Zimmerman (1978) stat- ed that turgor potential is partially or fully main- tained by osmoregulation during water stress by a reduction in the outflow of water from the cell. In previous study, the decrement of the leaf water Table 8 - Calcium content of mangosteen seedling in different water availability treatments at 2 months post-treatment Numbers, in the same column, followed by the same letter indicates no significant differences based on DMRT at a probability level of 5%. Treatment Calcium (%) Dissolved Pectate Phosphate Oxalate Total Young leaves Control (100%) 0.133 c 0.134 c 0.084 c 0.405 0.756 c CD (50%) 0.396 bc 0.247 bc 0.096 bc 0.547 1.287 bc CD (30%) 0.349 c 0.227 bc 0.091 c 0.428 1.096 bc PR1 (100%A:50%B) 0.473 bc 0.290 abc 0.135 ab 0.668 1.568 ab PR2 (100%A:30%B) 0.826 ab 0.429 ab 0.135 ab 0.372 1.763 ab PR3 (50%A:30%B) 1.065 a 0.488 a 0.157 a 0.425 2.136 a Mature leaves Control (100%) 0.322 b 0.267 b 0.124 b 0.611 1.326 b CD (50%) 0.741 a 0.462 a 0.319 a 0.814 2.337 a CD (30%) 0.600 ab 0.362 ab 0.187 ab 0.501 1.651 ab PR1 (100%A:50%B) 0.690 a 0.428 a 0.185 ab 0.743 2.047 ab PR2 (100%A:30%B) 0.814 a 0.346 ab 0.209 ab 0.68 2.051 ab PR3 (50%A:30%B) 0.763 a 0.441 a 0.174 ab 0.473 1.851 ab Branch Control (100%) 0.078 0.171 0.086 b 0.763 bc 1.099 bc CD (50%) 0.134 0.198 0.107 ab 0.923 abc 1.364 abc CD (30%) 0.144 0.159 0.104 ab 0.555 c 0.963 c PR1 (100%A:50%B) 0.156 0.247 0.127 ab 1.266 a 1.798 a PR2 (100%A:30%B) 0.142 0.236 0.139 a 1.108 ab 1.627 ab PR3 (50%A:30%B) 0.148 0.227 0.123 ab 1.335 a 1.835 a Root Control (100%) 0.202 b 0.373 0.148 0.675 1.399 CD (50%) 0.342 a 0.307 0.219 0.971 1.84 CD (30%) 0.231 ab 0.317 0.192 0.862 1.604 PR1 (100%A:50%B) 0.185 b 0.232 0.167 1.443 2.052 PR2 (100%A:30%B) 0.208 b 0.247 0.17 1.296 1.904 PR3 (50%A:30%B) 0.273 ab 0.287 0.198 1.316 2.075 Adv. Hort. Sci., 2018 32(1): 49-59 56 potential and relative water content were occurred in stressed wheat (Siddique et al., 2001) and Hibiscus rosa-sinensis (Egilla et al., 2005). The decreasing of photosynthesis and transpira- tion rate in stress mangosteen seedlings indicated that both moderate (50% FC) and severe (30% FC) stress conditions greatly affect mangosteen gas exchange. Purwanto and Agustono (2010) reported that photosynthesis rate in soybean, which was watered at a condition of 60% FC, decreased by 50%, while no significant fall in transpiration rate was noticed. As response to drought stress, stomata respond by reducing aperture, thereby restricting water loss, however, an inevitable consequence is the photosynthesis and canopy transpiration (Loveys et al., 1999) reduction. As mentioned in Table 6, control and PR1 treat- ments showed the longest roots compared to the other treatments, while the root volume did not show any differences among treatments. Hidayat (2005) reported in his research that mangosteen root has a seasonal growth where roots alternately grow with shoots. It expected lead to the slow response of mangosteen roots to drought stress. On the other hand, no significant differences were noticed in PR1 treatment in terms of root dry weight compared to control (Table 5). This was in agreement with Liu et al. (2006) which reported that PRD increased bio- mass allocation to roots. Promoting root growth under PRD has been reported in grapevine (Dry et al., 2000), therefore this has been considered as an advantage of PRD irrigation. Drought stress condition lead to high production of MDA which used as a stress indicator in the plant. High MDA content indicated that the lipid peroxida- tion rate, as the main effect of oxidative damage (Gill and Tuteja, 2010), was also high. Sofo et al. (2005) conclude that there is a direct correlation between MDA and drought stress, particularly at severe degrees of stress. In present research, the most severe case was observed in PR3 treatment which had highly MDA level that coincided with decrements in leaf water potential, photosynthesis and transpira- tion rates. Besides mangosteen, lipid peroxidation was also noticed in cucumber (Kubis et al., 2014), bean (Svetleva et al., 2012) and maize (Ti-da et al., 2006) within a stress condition. The accumulation of proline is a common response in plants to abiotic stress. Increasing proline is a plant response to adjust its osmotic potential (Slama et al., 2006) which has a strong relation with plant water potential. In addition to its role as an osmolyte for osmotic adjustment, proline contributes to stabilizing sub-cellular structures, scavenging free radicals, and buffering cellular redox potential under stress conditions (Ashraf and Foolad, 2007). Present research was in agreement with Omidi (2010) which stated that in canola plants, proline content increased twofold as a result of drought stress treat- ment. In the present study, the control treatment had the high GPX activity but did not show significantly differences among treatments, except PR3. It indi- cates that in normal condition, mangosteen seedlings have the high antioxidant activity, then became high- er when the stress condition occurs, such as PR3. Halušková et al. (2009) stated that different abiotic stresses may cause differences in the GPX activation. Miller et al. (2010) noticed that GPX is plant protec- tor against free radical. Previous studies by Sofo et al. (2005) and Aganchich et al. (2007) have reported up- regulation of the antioxidant defense system in young olive plants subjected to different degrees of water stress. Mangosteen seedlings in limited water media pro- duced secondary metabolites more than control, especially terpenoid and fatty acid groups. Varied antioxidant profile in different plant species is one of the principle reasons for the different adaptability and abiotic stress tolerance in plants (Jamali et al., 2016). Changes in composition and synthesis in drought stress condition were reported for group of terpenoid such as vitamin E (Gershenzon et al., 1978), an important antioxidant that protects the cell from free radical effects (Serbinova and Packer, 1994) and photosynthetic apparatus (Fryer, 1992) from oxidative damages. In the present research, the increasing in vitamin E content by 3.2%, in stress mangosteen seedlings, was a response to the stress condition. Abiotic stress-induced changes in the fatty acid composition of plant membrane lipids mainly occur through the regulated activities of fatty acid desaturases (Upchurch, 2008). Arabidopsis thaliana shows remarkable tolerance to drought stress and has capacities to maintain polar lipid content and sta- ble lipid composition, and increase the fatty acid unsaturation (Gigon et al., 2004). Mangosteen seedlings under PR1 treatment had the highest calcium level. According to Bell and Biddulph (1963) some plants absorb calcium based on their physiological demand which is sometimes not comparable to the transpiration rate. As men- Hapsari et al. - Partial root-zone irrigation 57 tioned in Table 8, calcium contents were higher in drought stress plants in treatments of CD1, PR1, PR2 and PR3. Allegedly, there was a relationship between the response of mangosteen seedlings and both long watering interval time and calcium metabolism. CD2 treatment had a longer watering interval time com- pared to CD1 so that when the drought signaling occurred the water was not available, leading to a fail in calcium absorption. The increment in calcium lev- els in CD1, PR1, PR2 and PR3 treatments occurred in the forms of Ca-pectate and Ca-dissolved, both of which play a role in cell wall component (Peaucelle et al., 2012) and cytoplasmic transduction signaling (Klimecka and Muszyńska, 2007), respectively. This was in agreement with Jin et al. (2016) which report- ed that increment in calcium levels in a salinity stress condition occurred in Ziziphus jujuba species. Mangosteen seedlings in PR1 treatment had higher water use efficiency (WUE) leading the possibility of calcium absorption in stress condition. In PR treat- ment, improvement in WUE was a results from par- tial stomatal closure. However, an inevitable conse- quence is the photosynthesis and canopy transpira- tion reduction (McCarthy et al., 2002). Hu et al. (2008) showed that PR method in maize able to pre- serve 29.5-33% water and increased WUE. The use of partial root-zone or deficit irrigation in grapevine (Vitis vinifera) increased WUE by about 40% while only decreasing yield by 15% when compared with full irrigation (Dos Santos et al., 2003). Mangosteen seedlings had a low response and different mechanisms in facing drought, by increasing MDA, proline, Ca-pectate and Ca-dissolved, GPX activity and synthetizing various secondary metabo- lites in mature leaves. In the present study, there were no significant differences between control and PR1 treatments in leaf morphology, proline, MDA and GPX activity, indicating that PR1 treatments were not severely water stressed. Therefore, present research concluded that irrigation of mangosteen through PR method was a promising method for implementation, especially in limited water areas, when the soil moisture level represents 50% (or more) of the field capacity. The implementation of PR method can reduce water requirement without sig- nificantly affecting the plant growth. It can reduce both time and labor requirements. However, improvements in the technical application to develop an effective procedure for field implementation and its relation to flowering and yellow latex of mangos- teen, are still needed as further studies. Acknowledgements The authors would like to thank the Ministry of Research, Technology and Higher Education for fund- ing and supporting the present research through PMDSU program, Batch II, in the fiscal year of 2016. References ABDURACHMAN A., HARYATI U., JUARSAH I., 2006 - Determination of soil water content by gravimetric method, pp. 131-142. - In: KURNIA U., F. AGUS, A. ADIMIHARDJA, and A. DARIAH (eds.) Soil physical prop- erties and its analysis method . Department of Agriculture, Jakarta, Indonesia, pp 282. [In Indonesian]. ADWIRMAN, 2006 - Effects of water stress on physiological and biochemical responses of mangosteen (Garcinia mangostana L.) plant. - Dissertation, University Putra Malaysia, Malaysia, pp. 147. AGANCHICH B., TAHI H., WAHBI S., ELMODAffAR C., SER- RAJ R., 2007 - Water relations, photosynthesis, growth and water use efficiency in tomato plants subjected to partial rootzone drying and regulated deficit irrigation. - Plant Biosyst., 141: 252-264. ASHRAF M., FOOLAD M.R., 2007 - Roles of glycine betaine and proline in improving plant abiotic stress resistance. - Environ. Exper. Bot., 59(2): 206-216. BATES L.S., 1973 - Rapid determination of free proline for water-stress studies. - Plant and Soil, 39: 205-207. BELL C.W., BIDDULPH O., 1963 - Translocation of calcium: exchange versus mass flow. - Plant Physiol., 38: 61-14. CHEN W., PROVART N.J., GLAZEBROOK J., KATAGIRI F., CHANG H.S., EULGEM T., MAUCH F., LUAN S., ZOU G., WHITHAM S.A., BUDWORTH P.R., TAO Y., XIE Z., CHEN X., LAM S., KREPS J.A., HARPER J.F., SI-AMMOUR A., MAUCH-MANI B., HEINLEIN M., KOBAYASHI K., HOHN T., DANGL J.L., WANG X., ZHU T., 2002 - Expression pro- file matrix of Arabidopsis transcription factor genes suggests their putative functions in response to envi- ronmental stresses. - Plant Cell, 14: 559-574. CHEN W.S., UETOMO S., 1976 - Studies on calcium absorp- tion in vegetable crops: the absorption and physiologi- cal significance of calcium in vegetative and reproduc- tive phase of plant growth. - J. Japan. Soc. Hort. Sci., 45: 33-42. DORLY SOEKISMAN T., JAIME A., SILVA T., POERWANTO R., EFENDI D., FEBRIYANTI B., 2011 - Calcium spray reduces yellow latex on mangosteen fruit (Garcinia Mangostana L.). - J. Fruit Ornam. Plant Res., 19(2): 51-65. DOS SANTOS T.P., LOPES C.M., RODRIGUES M.L., DE SOUZA C.R., MAROCO J.P., PEREIRA J.S., SILVA J.R., CHAVES M.M., 2003 - Partial root-zone drying: effects on growth and fruit quality of field-grown grapevines (Vitis vinifera). - Funct. Plant Biol., 30(6): 663-671. Adv. Hort. Sci., 2018 32(1): 49-59 58 DRY P.R., LOVEYS B.R., DURING H., 2000 - Partial drying of the rootzone of grape. 2. Changes in the patterns of root development. - Vitis, 39: 9-12. EGILLA J.N., DAVIES Jr F.T., BOUTTON T.W., 2005 - Drought stress influences leaf water content, photosynthesis, and water-use efficiency of Hibiscus rosa-sinensis at three potassium concentrations. - Photosynthetica, 43: 135-140. FRYER M.J., 1992 - The antioxidant effects of thylakoid vitamin E (α-tocopherol). - Plant Cell and Environ., 15: 381-392. GERSHENZON J., LINCOLN D.E., LANGENHEIM J.H., 1978 - The effect of moisture stress on monoterpenoid yield and composition in Satureja douglasii. - Biochemical Systematics and Ecology, 6: 33-43. GIGON A., MATOS A.-R., LAFFRAY D., ZUILY-FODIL Y., PHAM-THI A-T., 2004 - Effect of drought stress on lipid metabolism in the leaves of Arabidopsis thaliana (Ecotype Columbia). - Ann. Bot., 94: 345-351. GILL S.S., TUTEJA N., 2010 - Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. - Plant Phys. and Bioch., 48: 909-930. HALUŠKOVÁ L., VALENTOVIČOVÁ K., HUTTOVÁ J., MISTRÍK I., TAMÁS L., 2009 - Effect of abiotic stresses on glu- tathione peroxidase and glutathione S-transferase activity in barley root tips. - Plant Physiol. Bioch., 47 (11-12): 1069-1074. HIDAYAT R., 2005 - Study of dormancy period and growth rhythm in mangoosteen (Garcinia mangostana L.) shoot and root. - Bul. Agron., 33(2): 16-22. [In Indonesian]. HONG-BO S., LI-YE C., MING-AN S., 2008 - Calcium as a ver- satile plant signal transducer under soil water stress. - Bioessays, 30(7): 634-641. HU T., KANG S., LI F., ZHANG J., 2008 - Effects of partial root-zone irrigation on the nitrogen absorption and uti- lization of maize. - Agric. Water Manag., 96: 208-214. JAMALI B., ESHGHI S., KHOLDEBARIN B., 2016 - Antioxidant responses of ‘Selva’ strawberry as affected by salicylic acid under salt stress. - J. Berry Res., 6: 291-301. JIN J., CUI H., LV X., YANG Y., WANG Y., LU W., 2016 - Exogenous CaCl2 reduces salt stress in sour jujube by reducing Na+ and increasing K+, Ca2+, and Mg2+ in dif- ferent plant organs. - J. Hortic. Sci. Biotech., 92(1): 1-9. KLIMECKA M., MUSZYŃSKA G., 2007 - Structure and func- tions of plant calcium-dependent protein kinases. - Acta Biochimica Polonica, 54(2): 219-233. KORTH K.L., DOEGE S.J., PARK S.H., GOGGIN F.L., WANG Q., GOMEZ S.K., LIU G., JIA L., NAKATA A.P., 2006 - Medicago truncatula mutants demonstrate the role of plant calcium oxalate crystals as an effective defense against chewing insects. - Plant Physiol., 141: 188-195. KUBIS J., WIECZORE J.F., JELONEK M.A., 2014 - Polyamines induce adaptive responses in water deficit stressed cucumber roots. - J. Plant Res., 127: 151-158. KURNIADINATA O.F., 2015 - Role of calcium in overcome yellow latex in mangosteen fruit. - Dissertation, Bogor Agricultural University, Indonesia, pp. 102. [ In Indonesian]. KURNIAWATI A., POERWANTO R., SOBIR EFFENDI D., CAHYANA H., 2010 - Evaluation of fruit characters, xan- thones content, and antioxidant properties of various qualities of mangosteens (Garcinia mangostana L.). - J. Agron. Indonesia, 38(3): 232-237. LIU F., SHAHNAZARI A., ANDERSEN M.N., JACOBSEN S., JENSEN C.R., 2006 - Effects of deficit irrigation (DI) and partial root drying (PRD) on gas exchange, biomass partitioning, and water use efficiency in potato. - Scientia Horticulturae, 109(2): 113-117. LIU Q., KASUGA M., SAKUMA Y., ABE H., MIURA S., YAM- AGUCHI-SHINOZAKI K., SHINOZAKI K., 1998 - Two tran- scription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low tem- perature responsive gene expression, respectively, in Arabidopsis. - Plant Cell, 10: 1391-1406. LOVEYS B.R., DRY P.R., McCARTHY M.G., 1999 - Using plant physiology to improve the water use efficiency of horti- cultural crops. - Acta Horticulturae, 537: 187-199. MARTIAS MANSYAH E., 2014 - Strengthening mangosteen competitiveness, pp. 205-222. - In HARYONO, PASAN- DARAN E., K. SURADISASTRA, M. ARIANI, N. SUTRISNO, S. PRABAWATI, M.P. YUFDY, and A. HENDRIADI (eds.) Strengthening competitiveness of agricultural product. - IAARD Press, Jakarta, Indonesia, pp. 632. [In Indonesian]. MATRA D.D., POERWANTO R., SANTOSA E., SOBIR HIGASHIO H., ANZAI H., INOUE E., 2016 - Analysis of allelic diversity and genetic relationships among culti- vated mangosteen (Garcinia mangostana L.) in Java, Indonesia using microsatellite markers and morpholog- ical characters. - Tropical Plant Biol., 9(1): 29-41. McCARTHY M.G., LOVEYS B.R., DRY P.R., STOLL M., 2002 - Regulated deficit irrigation and partial root-zone drying as irrigation management techniques for grapevines. - FAO Water Reports, 22: 79-87. MILLER G., SUZUKI N., CIFTCI-YILMAZ S., MITTLER R., 2010 - Reactive oxygen species homeostasis and signaling during drought and salinity stresses. - Plant, Cell & Environ., 33(4): 453-467. MUNNE-BOSCH S., ALEGRE L., 2004 - Die and let live: leaf senescence contributes to plant survival under drought stress. - Funct. Plant Biol., 31: 203-216. MUSTAHA M.A., 2012 - Growth improvement of mangos- teen seedling by growing media modification. - Dissertation, Bogor Agricultural University, Indonesia, pp. 223. [In Indonesian]. OMIDI H., 2010 - Changes of proline content and activity of antioxidant enzymes in two canola genotype under drought stress. - Am. J. Plant Physiol., 5 (6): 338-349. OSMAN M.B., MILAN A.R., 2006 - Mangosteen - Garcinia mangostana. - Southampton Centre for Underutilised Crops, University of Southampton, Southampton, UK, pp. 170. PAULL R.E., NAKASONE H.Y., 1998 - Tropical fruits. - CAB International, Wallingford, UK, pp. 445. Hapsari et al. - Partial root-zone irrigation 59 PEAUCELLE A., BRAYBROOK S., HÖFTE H., 2012 - Cell wall mechanics and growth control in plants: the role of pectins revisited. - Front Plant Sci., 3: 121. PELEG Z., BLUMEWALD E., 2011 - Hormone balance and abiotic stress tolerance in crop plants. - Plant Biology, 14(3): 290-295. POERWANTO R., DORLY MAAD M., 2010 - Yellow latex of mangosteen. - Proc. of Perhorti, pp. 255-260. [In Indonesian]. PURWANTO, AGUSTONO T., 2010 - Study of soybean physi- ology in weed density variation in drought stress condi- tion. - J. Agroland., 17(2): 85-90. RAMLAN M.F., MAHMUD T.M.M., HASAN B.M., KARIM M.Z., 1992 - Studies on photosynthesis on young man- gosteen plants grown under several growth conditions. - Acta Horticulturae, 321: 482-489. SAURE M.C., 2005 - Calcium translocation to fleshy fruit: its mechanism and endogenous control. - J. Hort. Sci., 17: 65-85. SDOODEE S., CHIARAWIPA R., 2005 - Regulating irrigation during pre-harvest to avoid the incidence of translucent flesh disorder and gamboge disorder of mangosteen fruits. - Songklanakarin J. Sci. Technol., 27(5): 957-965. SDOODEE S., LIMPUN-UDOM S., 2002 - Effect of excess water on the incidence of translucent flesh disorder in mangosteen (Garcinia mangostana L.). - Acta Horticulturae, 575: 813-820. SEPASKHAH A.R., AHMADI S.H., 2010 - A review on partial root-zone drying irrigation. - Inter. J. Plant Prod., 4(4): 241-258. SERBINOVA E.A., PACKER L., 1994 - Antioxidant properties of α-tocopherol and α-tocotrienol. - Methods in Enzymology, 234: 354-366. SETYANINGRUM Y.I., 2011 - Yellow latex secretory anatomical response, morphology and physiology of mangosteen (Garcinia mangostana L.) to external calci- um application. - Thesis, Bogor Agricultural University, Indonesia, pp. 61. [In Indonesian]. SIDDIQUE M.R.B., HAMID A., ISLAM M.S., 2001 - Drought stress effects on water relations of wheat - Bot. Bull. Acad. Sinica, 41: 35-39. SLAMA I., MESSEDI D., GHNAYA T., SAVOURE A., ABDELLY C., 2006 - Effects of water deficit on growth and proline metabolism in Sesuvium portulacastrum. - Environ. Exper. Bot., 56: 231-238. SOFO A., DICHIO B., XILOYANNIS C., MASIA A., 2005 - Antioxidant defence in olive trees during drought stress: changes in activity of some antioxidant enzymes. - Funct. Plant Biol., 32: 45-53. SUWWAN M.A., POOVAIAH B.W., 1978 - Association between elemental content and fruit ripening in rin and normal tomatoes. - Plant Physiol., 13: 883-885. SVETLEVA D., KRASTEV V., DIMOVA D., MITROVSKA Z., MITEVA D., PARVANOVA P., CHANKOVA S., 2012 - Drought tolerance of Bulgarian common bean geno- types, characterised by some biochemical markers for oxidative stress. - J. Central Eur. Agr., 13: 349-361. TI-DA G., FANG-GONG S., LI-PING B., YIN-YAN L., GUANG- SHENG Z., 2006 - Effects of water stress on the protec- tive enzyme activities and lipid peroxidation in roots and leaves of summer maize. - Agric. Sci., 5: 101-105. UPCHURCH R.G., 2008 - Fatty acid unsaturation, mobiliza- tion, and regulation in the response of plants to stress. - Biotechnol. Lett., 30(6): 967-977. URBANEK H., KUZNIAK-GEBAROWSKA E., HERKA K., 1991 - Elicitation of defense responses in bean leaves by Botrytis cinerea polygalacturonase. - Acta Phys. Plant, 13: 43-50. WANG Y., DING M., GU X., WANG J., PANG Y., GAO L., XIA T., 2013 - Analysis of interfering substances in the mea- surement of malondialdehyde content in plant leaves. - Am. J. Biochem. Biotech., 9(3): 235-242. WIEBEL J., CHACKO E.K., DOWNTON W.J.S., LOVEYS B.S., LUDDERS P., 1994 - Carbohydrate levels and assimilate translocation in mangosteen (Garcinia mangostana L.). - Gartenbauwissenschaf, 60(2): 90-94. WIJAYANTO N., HARTOYO A.P.P., 2015 - Biodiversity based on agroforestry . - Proc. Indonesia Biodiversity Community, 1(2): 242-246. [In Indonesian]. WINGLER A., ROITSCH T., 2008 - Metabolic regulation of leaf senescence: interaction of sugar signaling with biot- ic and abiotic responses. - Plant Biology, 10(1): 50-62. ZIMMERMANN U., 1978 - Physics of turgor and osmoregu- lation. - Ann. Rev. Plant Physiol., 29: 121-148.