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coleoptile and leaf.
AGE AND ORGAN SPECIFIC ACCUMULATION OF ASCORBATE IN WHEAT (TRITICUM AESTIVUM L.) SEEDLINGS GROWN UNDER ETIOLATION ALONE AND IN COMBINATION WITH OXIDATIVE STRESS Amjad Hameed1 Nayyer Iqbal1 Salman Akbar Malik2 Hina Syed1 M. Ahsanul-Haq1 ABSTRACT Ascorbate plays a diverse role in plant growth and metabolism. The present study was planned to reveal out the dynamics and role of ascorbic acid during early ontogenesis of wheat seedlings grown under etiolation alone and combined with oxidative stress (100mM H2O2) condition. Ascorbic acid accumulates in all organs i.e. roots, leaf and coleoptile of wheat under both types of stress conditions. The accumulation was organ as well as age specific. For combined stressed seedlings, ascorbate contents were overlapping only in leaf and coleoptile and were significantly higher in roots on 5th, 7th and 8th day as compare to other organs. A highly significant organ specific deference in ascorbate contents was observed on 8th day in both types of seedlings. Roots maintained the maximum ascorbate followed by coleoptile and leaf. Combined stressed seedlings, as compared to etiolated seedlings, accumulated significantly higher ascorbic acid in leaf on 6thand 7th day and in roots on 5thand 7th day. Contrary to this ascorbic acid contents were significantly higher in coleoptile of etiolated seedlings on 8th day. Collectively it was obvious that ascorbic acid accumulation is essential for growth and development of both etiolated and combined stressed seedlings. The accumulation is organ and age specific being maximum in roots followed by
Keywords: etiolation, coleoptile, leaf, oxidative stress, roots, vitamin C, Wheat. List of abbreviations: APX= ascorbate peroxidase, ROS= reactive oxygen species, DCIP= 2,6-dichloroindophenol
RESUMO O ascorbato tem importância muito diversa no crescimento e metabolismo das plantas. O presente estudo foi planejado para revelar a dinâmica e a função do ácido ascórbico durante a ontogênese inicial de mudas de trigo crescidas sob estiolamento somente e combinadas com condição de estresse oxidativo (100mM H2O2). O ácido ascórbico acumulou-se em todos os órgãos isto é, raízes, folhas e coleóptilos de trigo em ambas as condições de estresse. O acúmulo foi tanto órgão como idade específico. Para plântulas de estresses combinados, o conteúdo em ascorbato foi sobreposto somente em folhas e coleóptilo e foi significativamente mais alto em raízes nos 5o, 7o e 8o dias quando comparados com os outros órgãos. Uma diferença órgão-específica altamente significante em conteúdo de ascorbato foi observada no 8o dia em ambos os tipos de plântulas. As raízes mantiveram o máximo de ascorbato, seguido por coleóptilo e folha. Plântulas de estresses combinados, quando comparados com plântulas estioladas, acumularam significantemente mais ácido ascórbico nas folhas nos dias 6 e 7 e em raízes nos dias 5 e 7. Ao contrário, a concentração de ácido ascórbico foi significativamente mais alta no coleóptilo de plântulas sob estiolação no dia 8. Coletivamente ficou óbvio que a acumulação de ácido ascórbico é essencial para o crescimento e desenvolvimento tanto de plântulas estioladas como de estresses combinados. O acúmulo é órgão e idade-específico sendo máximonas raízes, seguida do coleóptilo e das folhas. Palavras-chave: estiolamento, coleóptilo, estresse oxidativo, raízes, vitamina C, trigo.
________________ 1 Plant Molecular Breeding Group, Mutation Breeding Division, Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box. 128 Faisalabad, Pakistan.
[email protected] 2 Department of Biological Sciences, Qauid-i-Azam University, Islamabad, Pakistan. Caderno de Pesquisa Sér. Bio., Santa Cruz do Sul, v. 17, n. 1, p. 51-63, jan./jun . 2005
Caderno de Pesquisa Sér. Bio., Santa Cruz do Sul, v. 17, n. 1, p. 51-63, jan./jun . 2005
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INTRODUCTION
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MATERIALS AND METHODS
Ascorbate and related enzymes like ascorbate peroxidase play diverse roles in several physiological processes in plants, including growth, differentiation and metabolism (FOYER, 1993). Ascorbate is required for the progression of the cell cycle, cell elongation (MATAMOROS et al., 2003) and expansion (NOCTOR AND FOYER, 1998; SMIRNOFF and WHEELER, 2000). Ascorbate modulates cell growth by controlling the biosynthesis of hydroxyprolinerich proteins required for the progression of G1 and G2 phases of the cell cycle and redox reactions at the plasma membrane involved in elongation mechanisms (CORDOBA-PEDREGOSA et al., 1996). It also participates in the regulation of mitosis (NOCTOR and FOYER, 1998; SMIRNOFF and WHEELER, 2000). Ascorbate content not only act to regulate defense and survival but also modulate plant growth under optimal conditions by modify gene expression via phytohormones. Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling (PASTORI et al., 2003). Leaves often have as much ascorbate as chlorophyll but the extent of ascorbate accumulation depends on developmental and on environmental cues that may act via the rate of production of ROS (CONKLIN et al., 1996). Ascorbate regenerates the -tocopherol oxidized by reactive oxygen species (ROS) at the membrane-cytosol interface, is a direct scavenger of most ROS, and is the substrate of ascorbate peroxidase (APX) (MATAMOROS et al., 2003). In view of the multiple roles of ascorbate, changes in its concentration may have important consequences for cellular and metabolic regulation (VELJOVICJOVANOVIC et al., 2001). Possible role of ascorbate concentration in development and in response to the environment must be evaluated in order to separate ascorbate-mediated effects from those due to concomitant changes in other factors. Present study deals with the dynamics of ascorbic acid in early growth and development of different organs in etiolated wheat seedlings. We have also attempted to reveal that how oxidative stress induced by exogenous application of hydrogen peroxide modulate the ascorbate contents during early seedling development, and whether or not plant response age or organ specifically.
To induce etiolation stress uniform sized seeds (44.05± 3.07mg) of wheat strain 1076 (Triticum aestivum L.) were germinated in petridishes at 25±1°C on wet filter paper under complete darkness. For applying oxidative stress along with etiolation, water as the medium was changed with 100 mM hydrogen peroxide (Merck, Germany) after 48 hours of germination, while other growth conditions were kept constant. The reagent solution (100 mM H2O2) was changed once a day for freshly prepared solutions to ensure the continuity and uniformity of applied oxidative stress. To get the sample for analysis the seedlings of defined age (seedling age was estimated in days starting from the time of seed soaking) were thoroughly washed with sterile distilled water. Roots, initial leaf and coleoptile were separated and grounded with a mortar and pestle under chilled condition in homogenization buffer. The homogenate was centrifuged at 12000rpm for 10 min. The supernatant was separated and used for ascorbate estimation. Ascorbic Acid (Vitamin C): The method used for ascorbic acid determination was 2,6-dichloroindophenol (DCIP) method of the Association of Vitamin Chemists (1961), that measures only reduced ascorbic acid. Briefly, each molecule of vitamin C converts a molecule of DCIP into a molecule of DCIPH2, and that conversion can be monitored as a decrease in the absorbance at 520nm. A standard curve was prepared using a series of known ascorbic acid concentrations. A simple linear regression equation was calculated to find the ascorbate concentration in unknown samples. Statistical Analysis: All experiments were repeated three times, every time with three replications (12 seedlings per replication). Similar results and identical trends were obtained each time. The data being presented here is for one experiment replicated three times with 12 seedlings per replication for each day. The descriptive statistics were applied to analyze and organize the resulting data. The f-test was applied to find differences in variance among samples. The significance of differences between means (for etiolation alone and in combination with etiolation stress) for different parameters was measured using Student’s tTest (two tailed) assuming unequal variances at 0.01 and where applicable at 0.05 significance level. All the statistical calculations were performed using computer software Microsoft Excel 2000.
Caderno de Pesquisa Sér. Bio., Santa Cruz do Sul, v. 17, n. 1, p. 51-63, jan./jun . 2005
Caderno de Pesquisa Sér. Bio., Santa Cruz do Sul, v. 17, n. 1, p. 51-63, jan./jun . 2005
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Results We studied the dynamics of ascorbic acid contents during early ontogenesis in root, initial leaf and coleoptile of wheat seedlings growing under etiolation alone and in combination with oxidative stress induced by exogenous hydrogen peroxide. Ascorbic Acid in roots: An overall steady increase (Fig 1) in ascorbic acid concentration was observed after 4th day in roots of seedlings growing under etiolation stress. A highly significant (p
