TANGGAP FISIOLOGI TANAMAN LIDAH BUAYA TERHADAP CEKAMAN ALUMUNIUM DENGAN PEMBERIAN DERIVAT ASAM FENOLAT DAN KORBOKSILAT
( Aluminum-Induced Physiological Responses of Aloe vera Grown in the Presence of Phenolic and Carboxylic Acid Derivatives )
Hastin Ernawati Nur Chusnul Chotimah1*) , Sudirman Yahya2), Munif Ghulamahdi2), Supiandi Sabiham3)
1) Program Study of Agrotechnology Faculty of Agriculture Palangkaraya University
Jl. Yos. Sudarso Palangkaraya 73112 Central Kalimantan Indonesia, Telephone +62 536 3326196
2) Department of Agronomy and Horticulture Bogor Agricultural University 3) Department of Soil and Land Resources Bogor Agricultural University
*) e-mail : email@example.com
Percobaan ini bertujuan untuk mempelajari tanggap fisiologi tanaman lidah buaya terhadap cekaman aluminium (Al) dengan pemberian derivat asam fenolat dan karboksilat. Dekomposisi bahan organik menghasilkan beberapa senyawa kimia seperti asam alifatik, fenolik dan polimer komplek fenol. Penelitian menggunakan Rancangan Acak Lengkap (RAL) dengan 3 kali ulangan. Apabila tidak terjadi pengaruh yang ditunjukkan oleh analisis ragam (ANOVA) dilanjutkan uji kontras ortogonal. Hasil penelitian menunjukkan bahwa kelompok derivat asam karboksilat menyebabkan peningkatan akumulasi Al akar dan P akar. Di bagian tajuk, pemberian kelompok derivat asam karboksilat menyebabkan penurunan Al tajuk dibandingkan perlakuan asam lengkap minus derivat asam karboksilat. Pemberian derivat asam fenolat dan karboksilat menyebabkan tanaman lidah buaya mengakumulasi malat dan oksalat. Pita-pita protein yang terbentuk sebagai tanggap terhadap cekaman aluminium dan pemberian derivat asam fenolat dan karboksilat berada pada kisaran 7.48-139.41 kDa.
Kata kunci : Respon fisiologi, Aloe vera, keracunan aluminium, derivat asam fenolat dan karboksilat.
Toxic effects of aluminum (Al) on plant growth have been attributted to several physiological responses that related to ability of plants to produce certain organic compounds. Decomposition of organic matter provides a number of biochemical compounds such as aliphatic acids, phenols, phenolic acids and complex polymeric phenols. The aim of this research was to study aluminum-induced physiological responses of Aloe vera grown in the presence of phenolic and carboxilyc acid derivatives. Research was conducted at the green house of Soil Research Institute, Laladon, Bogor. Experiment was arranged on Complete Randomized Design with three replications. Contrast orthogonal test was performed whenever there was a significant effect of the treatment as shown by ANOVA. Application of carboxylic acid derivatives group increased Al root and P root accumulation, while at the top, carboxylic acid derivatives group decreased Al shoot in comparison to group of complete acids minus carboxylic acid derivatives. Aloe vera also accumulated malate and oxalate as chelating agents on the tolerance mechanism to Al. In Aloe vera root protein profile, we demonstrated the specific protein with molecular weights ranging from 7.48 to 139.41 kDa.
Key words : Physiological responses, Aloe vera, aluminum toxicity, phenolic and carboxylic acid derivatives
Aloe vera is one commodity that has a chance to be developed in the tropics. At this time the aloe vera plant development is mostly done on peat soil, while the aloe vera plant development in the mineral soil faces such constraints as the low acidic soil acidity, poor soil organic matter and high content of Al.
Toxicity of aluminum (Al) becomes the main factor limiting crop productivity on acid soils because Al3+ cations at micromolar concentrations can be toxic to plants and inhibit growth. Al has a high affinity or bind strongly to a number of proteins, inorganic phosphate, nucleotides, RNA, DNA, carboxylic acids, phospholipids, flavonoids, anthocyanins, and peroxidation of fat. Mitochondrial activity was also reported to be hampered, followed by inhibition of respiration and reduced ATP (Rout et al. 2001; Ma et al. 2001; Samac and Tesfaye, 2003; Pineros et al. 2005; Ryan et al. 2009).
Peat as well as a stretch of land is also an organic material.
Decomposition of organic matter in flooded conditions will produce a lot of organic acid derivatives containing phenolic acids and carboxylic acid (PCA).
PCA derivatives have a functional group containing oxygen, a reactive site in the binding of metals, including Al and Fe. Thus, the activities of Al and Fe ions which are toxic to plants is reduced (Gerke, 1999). The result of decomposition of organic material in the form of organic acids will disable the phosphate binder; namely, Al and Fe through the formation of complex metal-organic compounds (Suriadikarta et al. 2002).
Some research results summarized by Hiradate and Yamaguci (2003) showed that the concentration of Al13 in solution is reduced by the presence of silisilat acid, sulfate and organic ligands such as tartrate, acetate, oxalate, lactate, salicylate and several low molecular weight phenolic ligand. Meanwhile, Yang et al. (2003) reported that salicylic acid can suppress the growth inhibition by Al on Cassia tora L to secrete more citric acid than the control.
Laboratory studies using organic acids from the group of mono-, di- and tri-carboxylic showed that the tri-carboxylic groups were able to increase the release of P from phosphate fertilizer by 57 mmol of P per kilogram, the-carboxylic group of 54.2 mmol per kilogram, and the group mono-carboxylate by 9.21 mmol of P per kilogram (Kpomblekou-A and Tabatabai, 2003). Meanwhile, Pang et al. (2007) reported that in barley plants, the influence of mono-carboxylic groups (format, acetate and propionate) caused the absorption of K+ ions stronger than the phenolic group (benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic). Phenolic acid caused increased absorption of Ca2+, whereas mono-carboxylic groups caused the release of Ca2+ ions from the root.
On the nucleic acid content and protein synthesis, Baziramakenga et al. (1999) reported that benzoic acid, cinnamic, vanillic and ferulic lowered 32P uptake, whereas p-hydroxybenzoic acid and p-coumaric acids increased the absorption of 32P in soybean seedling roots. At a concentration of 250 μM, all tested acid (benzoic acid, p-hydroxybenzoic, vanillic, cinnamic, p-coumaric and ferulic) reduced absorption 32P associated with the synthesis of DNA and RNA. At a concentration of 250 μM, all tested acid (benzoic acid, p-hydroxybenzoic, vanillic, cinnamic, p-kumarat and ferulat) reduced absorption of 32P associated with the synthesis of DNA and RNA. Benzoic acid, cinnamic, ferulat, vanillic, also caused a decrease in 35S-methionine uptake, whereas p-hydroxybenzoic acid and p-coumaric were able to increase the uptake of 35S-methionine. At a concentration of 125 μM, except p-coumaric acid and vanillic, all tested phenolic acids significantly decreased the content of 35S-methionine protein synthesis associated with soybean seedling roots.
This research aims to study the effect of organic acids that have -COOH and phenolic groups on aluminum which is represented in the study of plant physiology. Phenolic and carboxylic acid derivatives used are based on Chotimah et al. (2008), namely p-coumaric, p-hydroxybenzoic, sinapic, siringic, acetic, propionic, butyric, and succinic.
MATERIAL AND METHODS
Time and Place of Research
Experiments were conducted in Greenhouse Soil Research Institute Laladon Bogor. Analysis of P and Al content of root and shoot was carried out at the Soil Research Institute Laladon Bogor, analysis of protein profiles in the Laboratory of Biochemistry Bogor Agriculture University, while the determination of citrate, malate and oxalate was performed at the Integrated Laboratory of Faculty of Agriculture, Bogor Agriculture University. The planting material size was ± 20 cm taken from the separation of tillers.
Experimental design. This research used completely randomized design (CRD) with one factor, namely phenolic and carboxylic acid derivatives PCAD (O). PCAD factor as a treatment consists of 18 levels with three replications, so there were 54 experimental units. The levels of treatment were : O1 = + Aluminum (Al), O2 = p-coumaric acid, O3 = Al + p-hydroxybenzoic acid, O4 = Al + sinapic acid, O5 = Al + syringic acid, O6 = Al + acetic acid, O7 = Al + butyric acid, O8 = Al + propionic acid, O9 = Al + succinic acid, O10 = Al + complete acid (p- coumaric, p-hydroxybenzoic, sinapic, syringic, acetic, butyric, propionic, succinic), O11 = Al + complete acid – p-coumaric, O12 = Al + complete acid – p-hydroxybenzoic, O13 = Al + complete acid – sinapic, O14 = Al + complete – syringic, O15= Al + complete acid – acetic, O16 = Al + complete acid – butyric, O17 = Al + complete acid – propionic, O18 = Al + complete acid – succinic.
Preparation of root samples and treatments. Before treated, plants were adapted to provide samples of roots that will be used for further analysis. Experiments were conducted on nutrient solution with composition: A = 1.5 mM Ca (NO3)2.4H2O; 1.0 mM NH4NO3 ; 1.0 mM KCl ; 0.4 mM MgSO4.7H2O ; 2.0 mM KH2PO4. B = 0.50 ppm MnS04.H20 ; 0.02 ppm CuS04 .5H20; 0.05 ppm ZnSO4. 7H2O ; 0.50 ppm H3BO3 ; 0.01 ppm NH6Mo7O24.4H2O. C = (5.57 g FeSO4.7H2O + 7.45 g Na2EDTA)/liter. D = AlCl3.6H2O. E = 1.00 N HCl ; 1.00 N NaOH (Kasim, 2001) modified. Implementation of research was initiated with the preparation of nutrient stock solution by mixing the nutrients A, B and C in a drum capacity of 250 liters. Before use, roots were soaked in a solution of Dithane and insecticide Decis for 20 minutes. After it was rinsed thoroughly, seedlings were then grown hydroponically on stiorofoam. Treatment adaptation was done for ± 6 months until roots and root hair branches grow. Replacement of nutrient solution was carried out once a week and during the experiment nutrient solution was flowed through the air using the aerator.
After 6 months of adaptation, the treatment with PCAD was performed. Preparation of stock nutrient solution containing AlCl3.6H2O (nutrient D) and the stock solution containing various PCAD was made before treatment. The composition of the nutrient used was 2.5 parts. Concentrations of Al and PCAD were based on Chotimah et al. (2008), which was given for 1854 ppm Al in the forms of AlCl3.6H2O and PCAD; they were : p-coumaric 1.675 ppm, p-hydroxybenzoic 1.84 ppm; sinapic 2.445 ppm; syringic 1.925 ppm (phenolic group); acetic 8.945 ppm; propionic 2.685 ppm: 3.675 ppm butyric, and succinic 1.98 ppm (carboxylic group). Plants were planted in a bottle with a capacity of 250 ml, then placed in a plastic bucket with a capacity of 2 liters. The treatment was carried out for 72 hours and every day pH was adjusted to 4.5. pH regulation was carried out using a portable digital pH meter by adding a 1:00 N HCl or 1.00 N NaOH (nutrient E) using a pipette. Replacement of nutrient solution was conducted every day (24 hours) and during the experiment, and nutrient solution was flowed by air using the aerator such as the media adaptation. SimakBaca secara fonetik
Plant growth. Weight of fresh, dry root, and stem was observed after treatment.
Al and P roots and shoot. Roots and plant shoot 0:25 g were dissolved using a mixture of HNO3 and HClO4–. Aluminum content of roots was measured using AAS using mixture of N2O and acetylene flame. P was determined using a Spectrophotometer at a wavelength of 639 nm.
Root protein profiles. Root protein profiles were determined by the method of SDS-PAGE 1 D. Weighed roots were crushed on a porcelain placed on top of ice cubes and added with 0.1 M Tris HCl pH 7 as much as 1.5 ml. Slurry was inserted to Eppendorf tube. Protein quantification was performed using a spectrophotometer with a wavelength of 339 nm. Protein extracts were then separated by acrylamide gel. Electrophoresis was run using a buffer solution electrode base, glycine and SDS pH 8.3 for 90 minutes. Gel electrophoresis results were stained with silver staining. Marker proteins used were Low Molecular Weight (LMW) 14400-97000 Dalton from Merck Germany.
Accumulation of oxalate, citrate and malate roots. The roots were washed and crushed using distilled water on a porcelain plate. Slurry of root was centrifuged 15.000 rpm for 30 minutes. Supernatant was taken and recentrifuged with the same speed and time, then injected into High Pressure Liquid Chromatography (HPLC). The HPLC conditions used were: UV detector 210 nm, an organic acid column, H2SO4 0.25 ml / liter mobile phase, 0.5 ml / min mobile phase flow rate ,and 40 oC column temperature .
Data analysis. The data were analyzed using analysis of varian (ANOVA) 5% and when there is a treatment effect, it is followed by orthogonal contrast test.
RESULTS AND DISCUSSION
Al and P content of the root shoot. Orthogonal contrast test (Table 1) showed that the carboxylic acid derivatives (CAD) (O6-O9) significantly increased the accumulation of Al roots than controls (O1), group of phenolic acid derivatives (PAD) (O2-O5), and complete acid group (CA)-CAD (O15-O18). However, the increase of Al roots by CAD group did not adversely affect the P roots. Treatment of CAD actually showed the highest root P accumulation, followed by treatment with CA (O10), the group CA-CAD and the PAD group.
In the shoot, CA-CAD treatment increased the Al canopy more significantly than CAD as a single treatment (O6-O9) and CA treatment, whereas on the P shoot, CA increased Al shoot more real than the group of CA-CAD. The group of CAD increased more P shoot when compared to the PAD. The CAD influence was more pronounced when given singly (O6-O9).
Higher Al accumulation in roots due to the CAD treatment showed that the group of CAD was able to react with Al cations rapidly to form a chelate. Stable chelate prevents the absorption of Al by the cells and protect cells from the toxicity of Al so that the high accumulation of Al roots did not negatively affect the P roots.
Mechanism of Al chelation seems to occur when Al was entering the root tissue, so that Al stayed in the roots and caused less Al containing transpiration stream. Accordingly, the CA treatment produced the highest P shoot (Table 1). According to Sauvant et al. (1999), cellular response to Al as a result of humic substances and phenolic compounds showed that cell toxicity increased when there were chelate of Al with citric, salicylate, vanilic, p-coumaric and 4-hydroxyasetofenon. Meanwhile, between Al with humic chelates, EDTA, cafeic and protocatecuic protected cells against the toxicity of Al.
The concentration of oxalate and malate roots. Further analysis (Table 1) showed that the CAD was not able to increase root oxalate compared to controls (O6-O9 vs O1) as well as the CA-group CAD (O15-O18 vs O1), but the CA-CAD groups were more positive towards the oxalate roots than the treatment of CAD individually. On the concentration of malate, giving CA (O10) significantly increased the accumulation of malate in the roots compared to controls (O1) and treatment of other groups (O2-O5), (O6-O9), (O11-O14) and (O15-O18) .
Al-induced organic acid transport system was identified as an anion channel. These channels are membrane-bound proteins whose presence was activated by aluminum in the plasma membrane (Ma et al. 2001). Providing CA significantly produced the highest concentration of malate, which means that the treatment of CA plasma membrane was more permeable to malate than oxalate in roots of Aloe vera.
Malate is an organic acid that plays a role in Al tolerance mechanism externally and internally. In addition, malate involves in important processes of plant physiology. According to Schulze et al. (2002), malate contributed to the process of respiration, photosynthesis, fatty acid oxidation, lignin biosynthesis, the function of stomata, nitrogen fixation, amino acid biosynthesis, ion balance, uptake of P and Fe as well as tolerance to aluminum.
Root protein profiles. Protein test (Figure 1) using 1D SDS PAGE showed that the protein bands were formed in the range of 7.48-139.41 kD. The band of protein occurred the most in the treatment of Al + succinic acid (S9) with a molecular weight of 7.48-139.41 kD, whereas the protein bands appeared occurred the least in the treatment of p-coumaric acid (S2) with a molecular weight of 11.42-24.09 kD. According Sabehat et al. (1999), proteins in this range include Heat Shock Proteins (HSP) proteins with low to high molecular weight. HSP proteins in the range of 60-110 kDa is a high molecular weight proteins and 15-45 kD as low molecular weight. HSP protein is known as a protein that plays a role in the mechanisms of plant adaptation to face environmental stress. Meanwhile, Dubey and Pessarakli (1999) reported that metals stress would induce or inhibit the action of the enzyme, and induced the synthesis of cysteine-rich polypeptides binding to metals. This polypeptide served to suppress the presence of metal ions.
- Toxicity of Al can be reduced by giving phenolic and carboxylic acid derivatives from carboxylic acid derivatives group by forming chelate between carboxylic acid derivatives and Al cations.
- Aloe vera plants accumulate oxalate and malate acid in response to aluminum and the applying of phenolic and carboxylic acid derivatives.
- Bands of proteins is formed on the roots of aloe vera as a response to aluminum and phenolic and carboxylic acid derivatives including of Heat Shock Proteins (HSP) in the molecular weight range of 7.48-139.41 kDa
The author would like to thank to Ditjen Dikti Kemendiknas (Directorate General for Higher Education, National Education Ministry) under the Post Graduate Student Scholarships and Indonesia Toray Science Foundation for granting financial support that made this project possible to be conducted.
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S11=Al+ complete acid -p- coumaric
S12=Al+ complete acid –p-hydroxybenzoic
S13=Al+ complete acid – sinapic
S14=Al+ complete acid – siringic
S15=Al+ complete acid – acetic
S16=Al+ complete acid – butyric
S17=Al+ complete acid – propionic
S18=Al+ complete acid – succinic
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