Farklı arpa (Hordeum vulgare L.) çeşitlerinde alüminyum toksisitesi etkilerinin fizyolojik ve biyokimyasal olarak incelenmesi
Physiological and biochemical investigation of the effects of aluminium toxicity in different barley (Hordeum vulgare L.) genotypes
Yazar: MERVE YILMAZ KAÇAR
Danışman: DR. ÖĞR. ÜYESİ ALİ DOĞRU
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Biyoloji = Biology

Özet:
Bu çalışmada farklı arpa (Hordeum vulgare L.) genotiplerinde (Çetin-2000 ve Zeynelağa) alüminyum (Al2O12S3.18H2O; 3 mM) toksisitesinin etkileri erken fide evresinde fizyolojik, biyokimyasal ve fotokimyasal olarak araştırılmıştır.
Al toksisitesi iki arpa genotipinde de kök ve gövde büyümesini yavaşlatmış ancak büyüme hızı üzerindeki inhibisyon etkisinin köklerde daha belirgin olduğu görülmüştür. Taze ağırlık bakımından gövdelerin Al toksisitesine daha duyarlı olduğu, kuru ağırlık bakımından ise Al toksisitesinin kök üzerinde daha etkili olduğu gözlenmiştir. Al toksisitesi Çetin-2000 genotipinin yapraklarındaki oransal su miktarını azaltmış, Zeynelağa genotipinde ise değiştirmemiştir. Bu sonuç Al toksisitesinin Çetin-2000 genotipinde su ilişkilerini bozduğunu göstermektedir. Her iki arpa genotipinin yapraklarındaki membran stabilite indeksi Al toksisitesinden etkilenmemiştir. Al toksisitesi altındaki köklerde formazon miktarı ve Evan's mavisi alınımında meydana gelen değişimler Çetin-2000 genotipinde kök metabolizmasının Al toksisitesine daha duyarlı olduğunu göstermiştir. Al stresi altındaki Çetin-2000 genotipinin yapraklarındaki antosiyanin miktarı kontrolle karşılaştırıldığında önemli derecede artmıştır. Ancak Zeynelağa genotipinde belirgin derecede azalmıştır. Bu da Zeynelağa genotipinde Al toksisitesine karşı savunma mekanizmasının bozulduğunu gösteriyor olabilir. Çetin-2000 genotipinin yapraklarındaki fotosentetik pigment (klorofil a, klorofil b, toplam klorofil ve toplam karotenoid) miktarı Al toksisitesi sonucu kontrolle karşılaştırıldığında önemli derecede azalmıştır. Bu sonuç Al toksisitesi altındaki Çetin-2000 genotipinde fotosentetik pigmentlerin parçalanma hızının artmasından ve/veya sentez hızının azalmasından kaynaklanıyor olabilir. Al toksisitesi altındaki Zeynelağa genotipinde ise fotosentetik pigment miktarı değişmemiştir. Klorofil a floresansı ölçümleri ve JIP testi parametrelerindeki değişimler Al toksisitesi sonucu her iki genotipte de fotosentetik elektron taşınım reaksiyonlarının olumsuz yönde etkilendiğini göstermiştir. Bunun dışında Al toksisitesinin her iki arpa genotipinde de fotosistem II birimlerini yapısal ve fonksiyonel anlamda etkilediği söylenebilir. Ancak diğer analiz sonuçları ile birlikte değerlendirildiğinde, Çetin-2000 genotipinde fotosentetik aktivitedeki değişimler, bu genotipin Al toksisitesine uyum sağlayabildiğini göstermiştir. Zeynelağa genotipi ise Al toksisitesinden fotokimyasal olarak zarar görmüştür.
Sonuç olarak, Çetin-2000 genotipinin Al toksisitesine daha toleranslı olduğu ve Al toksisitesine uyum göstermesini sağlayan daha elastik bir mekanizmaya sahip olduğu söylenebilir. Ancak Zeynelağa genotipi Al toksisitesinden metabolik olarak zarar görmüştür.
Anahtar kelimeler: Al toksisitesi, arpa, klorofil a floresansı, JIP testi, fotosentetik aktivite

Abstract:
In this study, the physiological, biochemical and photochemical effects of aluminium (Al2O12S3.18H2O; 3 mM) toxicity in some barley (Hordeum vulgare L.) genotypes (Çetin-2000 and Zeynelağa) has been investigated at the early seedling stage.
Seeds that are used in this study were obtained from the Republic of Türkiye Ministry of Agriculture and Forestry Field Crops Central Research Institute, Ankara, Türkiye. After sterilized in 5% sodium hypochlorite for 10 minutes, barley seeds were imbibed in bidistilled water for 4 hours, placed between wet filter papers in plastic bags and allowed to germinate in climate chamber at 25 C in a dark environment for 3 days. Three days of uniform seedlings were transferred to the plastic pots containing perlite and Hoagland's nutrient solution and kept in a climate room under 18/25 C temperature (night/day), 16/8 hours photoperiod (day/night), 50±5% relative humidity and 200 µmol photon m-1 s-1 light intensity for 10 days. Plants were exposed to aluminium toxicity (3 mM, Al2O12S3.18H2O) for 5 days, while control plants were watered with Hoagland's nutrient solution until at the end of the experiment. After measuring chlorophyll a fluorescence in the intact leaves, plants were harvested.
The root and shoot length of barley plants were measured with a milimetric ruler. The fresh weight of roots and shoots were separately weighted and plant materials were kept in an oven at 70 C for 3 days until the constant weight was reached. After that the dry weight of root and shoot of barley plants were determined. The leaf samples were immediately used to determine relative water content, membrane stability index, anthocyanin, and photosynthetic pigment content. Dehydrogenase activity was measured and Evan's blue test was done in the root samples. Experiments were a randomized complete block design with different independent replicates. Analysis of variance (ANOVA) was performed using SPSS 20.0 statistical software for Windows. To separate significant differences between means, Duncan test was used at P = 0.05.
Al toxicity decreased root growth in both barley genotypes, probably due to direct contact of roots with Al-rich nutrient solution. But the rate of growth inhibition in roots of the genotype Çetin-2000 was found to be more remarkable. The sensitivity of the root and shoot growth to Al toxicity in the genotype Çetin-2000 was almost the same, while shoot growth in the genotype Zeynelağa was less inhibited by Al toxicity compared to the genotype Çetin-2000. These results may show that transport of Al ions from roots to shoots in the genotype Zeynalağa was restricted by some metabolic mechanisms and/or physical barriers. Root fresh weight in the genotype Çetin-2000 was not significantly affected by Al toxicity, while it was remarkably decreased in the genotype Zeynalağa as a result of excess Al application. Biomass accumulation in roots of both barley genotypes, however, was dramatically declined by Al toxicity as indicated by the lowered dry weight. Al application caused significant decreases in shoot fresh weight of both barley genotypes. Shoot dry weight in the genotype Zeynelağa, on the other hand, was not significantly affected by Al toxicity while it was remarkably diminished in the genotype Çetin-2000. These results clearly show that inhibitory effect of Al application on biosynthetic capacity in roots of both barley genotypes was more remarkable compared to shoots. Relative water content in the leaves of the genotype Çetin-2000 was significantly reduced by Al toxicity, which shows that Al toxicity interfered with water relations in this genotype. However, it was not affected by Al application in the genotype Zeynalağa. Al toxicity did not lead to membrane damage in the leaves of both barley genotypes as shown by the constant membrane stability index compared to corresponding controls. Formazon content in the roots of both barley genotypes under Al toxicity was found to be drastically lower than respective controls. However, formazon content in the roots of the genotype Çetin-2000 was more severely decreased by Al toxicity, indicating the fact that reactions of the cellular respiration in roots of this genotype was inhibited to a greater extent compared to the genotype Zeynelağa. Evan's blue content in roots of the genotype Çetin-2000 was significantly lower than the respective control, while it was not significantly affected by Al application in the genotype Zeynelağa. Similarly, these results also showed that vitality of the root tissue was drastically disrupted in the genotype Çetin-2000 under Al toxicity. Anthocyanin content in the leaves of the genotype Çetin-2000 was significantly increased by Al toxicity whereas it was drastically lower than the respective control in the leaves of the genotype Zeynelağa. It is obvious that the genotype Çetin-2000 could represent higher capacity to resist Al toxicity through anthocyanin accumulation in leaf tissues. However, the decreased level of anthocyanin in the leaves of the genotype Zeynelağa may show that either this barley genotype is sensitive to Al toxicity or the Al concentration used in this study is too low to affect this genotype metabolically. Chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoid content in the leaves of the genotype Çetin-2000 under Al toxicity was remarkably diminished compared to the respective control, while these parameters remained unchanged in the leaves of the genotype Zeynelağa as a result of Al application. In the case of the genotype Çetin-2000, the decreased level of photosynthetic pigment content in leaves may be explained by the accelerated rate of destruction and/or the slowed down of synthesis of the photosynthetic pigments under Al toxicity. On the other hand,  this may be a kind of strategy for the genotype Çetin-2000 to reduce photosynthetic pigment pool to provide an equilibrium between the absorbed and photochemically quenched light energy under the condition of Al toxicity.
Changes in the chlorophyll a fluorescence measurements and JIP test parameters showed that photosynthetic electron transport reactions were impaired in the genotypes Çetin-2000 and Zeynelağa as a result of Al toxicity. Fo (minimum fluorescence), for example, was significantly decreased in the leaves of the genotype Çetin-2000 under Al toxicity, while it was drastically increased by excess Al application in the genotype Zeynelağa. These results show that Al toxicity led to the decreased transport rate of the light energy from the pigment molecules to the reaction centres or conversion of the reaction centres from active to inactive form. Another possible explanation for the increased Fo value may be that Al toxicity inhibited the movement of electrons from quinone A to quinone B, and hence decreased the ability of the photosystem II units to trap light energy effectively. Fm (maximum fluorescence) in the leaves of the genotype Çetin-2000 was found to be significantly lower than the respective control, indicating that the stability of the acceptor side of the photosystem II units was diminished by Al toxicity. Fv/Fm (maximum quantum efficiency of photosystem II), which must be around 0,83 under optimum conditions, was significantly lower than control in the genotype Zeynelağa under Al toxicity, while it remained unchanged in the genotype Çetin-2000, indicating that Al toxicity led to a remarkable photoinhibition in photosystem II activity in the genotype Zeynelağa. Also, the decreased φPo value in the genotype Zeynelağa under Al toxicity as compared to the genotype Çetin-2000 showed that the primary photochemical reactions in the genotype Zeynelağa was inhibited to some extent. In this study, the parameters area, Fv/Fo, and V/to in both barley genotypes under Al toxicity did not represent significant changes compared to the respective controls. However, the lower level of area in the genotype Çetin-2000 under Al toxicity may indicate the decreased rate of electron transport which has a regulatory effect on photosystem II activity as compared to the genotype Zeynelağa devoiding of this mechanism. Compared to the genotype Çetin-2000, significantly lower and higher values of Fv/Fo and V/to in the genotype Zeynelağa respectively, showed that Al toxicity led to the inhibition of the efficiency of the Hill reaction and the accelerated rate of the accumulation of the closed reaction centres. Al toxicity significantly reduced o in the genotype Çetin-2000 and did not affect in the genotype Zeynelağa compared to the respective controls, indicating the restricted ability of an absorbed photon to move electrons from quinone A to the electron transport system in the genotype Çetin-2000. In this study, φEo and φDo in the genotypes Çetin-2000 and Zeynelağa under Al stress were not drastically affected compared to the corresponding controls. However, Al application led to the significantly lower φEo and higher φDo in the genotype Zeynelağa, respectively, compared to the Al-stressed Çetin-2000. These results showed that quantum efficiency of the electron transport from quinone A to plastoquinone was diminished, while quantum efficiency of thermal energy dissipation was accelerated in the genotype Zeynelağa under Al toxicity. Changes in the parameters ETo/RC and DIo/RC in both barley genotypes under Al stress also confirmed this fact. TRo/RC in both barley genotypes was not significantly affected by Al toxicity compared to the respective controls. However, the genotype Zeynelağa represented remarkably higher TRo/RC value as a result of Al application compared to the genotype Çetin-2000. This result indicated that maximum energy flux per reaction centre leading to the reduction of quinone A was increased in the genotype Zeynelağa compared to the genotype Çetin-2000. In addition, it has been found that Al toxicity negatively affected the structure and function of the photosystem II units in the genotype Zeynelağa compared to the genotype Çetin-2000. Taken together, changes in the genotype Çetin-2000 in terms of the photosynthetic activity indicated that this genotype could acclimate to Al toxicity. The genotype Zeynelağa, on the other hand, suffered from Al toxicity photochemically.
As a result, it may be concluded that the genotype Çetin-2000 could be more tolerant to Al toxicity and have an elastic metabolism to acclimate Al toxicity. In contrast, the metabolism of the genotype Zeynelağa was impaired by Al toxicity.
Key words: Al toxicity, barley, chlorophyll a fluorescence, JIP test, photosynthetic activity