A Potential Common Pyrroline-5-Carboxylate Synthetase (P5CS) Gene As A Stress Marker For qRT-PCR In Legume Plants

Abstract

Leguminosae (or Fabaceae), commonly known as legumes and including plants of great agricultural and ecological importance, are important agricultural plant families with a very wide distribution worldwide. Stress is one of the main environmental factors affecting the growth and productivity of cultivated plants. Plants are designed to minimize the impact of stress and to stress resistance at the biochemical level by producing osmolytes such as proline to protect its components. Pyrroline-5-carboxylate synthetase (P5CS) is a key enzyme in the proline biosynthesis pathway in plants and is involved in the initial step of proline production. In this study, a common primer was identified to enable qRT-PCR-based studies as an alternative to biochemical methods for determining the increase in proline content. With this aim, important agricultural plants, Phaseolus vulgaris, Lens culinaris, Cicer arietinum, Glycine max, Pisum sativum, Medicago sativa, were exposed to three different stresses (drought, heat, salt). Proline amounts of the samples were determined biochemically and then gene expression levels were calculated using the common primer. In our results, proline levels were significantly increased in all plant samples. Relative gene expression analysis results also correlated with the biochemical results. In conclusion, this study revealed the existence of a common marker that can be used to determine proline levels at the gene expression level, which is an important marker indicating that some agricultural legume plants are under stress. Our findings show that investigating the expression of genes involved in proline biosynthesis can help to understand the stress responses of plants at the molecular level. In addition, analysis, especially by molecular methods, provides faster and more precise results. This allows to observe the dynamic changes of genes active in proline biosynthesis in a shorter time and changes at the gene level allow us to observe the plant's readiness or early responses to stress.

Keywords

• Leguminosae
• Abiotic stress
• Proline biosythesis
• Pyrroline-5-carboxylate synthetase (P5CS)
• Gene expression

Baklagil Bitkilerinde qRT-PCR İçin Stres Belirteci Olarak Potansiyel Ortak Bir Pirolin-5-Karboksilat Sentetaz (P5CS) Geni

Öz

Genellikle baklagiller olarak bilinen ve büyük tarımsal ve ekolojik öneme sahip bitkileri içeren Leguminosae (veya Fabaceae), dünya çapında çok geniş bir dağılıma sahip önemli tarım bitkisi familyasıdır. Stres, tarımı yapılan bitkilerin büyümesini ve verimliliğini etkileyen ana çevresel faktörlerden biridir. Bitkiler, stresin etkisini en aza indirmek ve hücrenin çeşitli bileşenlerini korumak adına prolin gibi osmolitler üreterek biyokimyasal düzeyde strese direnç göstermektedir. Pyrroline-5-carboxylate synthetase (P5CS) bitkilerde prolin biyosentezi yolunda kilit enzimdir ve prolin üretiminin başlangıç aşamasında yer alır. Bu çalışmada prolin miktarındaki artışın belirlenmesinde biyokimyasal olarak kullanılan yöntemlere alternatif qRT-PCR temelli çalışmalara olanak sağlayacak ortak bir primer belirlenmiştir. Bu hedefle önemli tarım bitkileri (Phaseolus vulgaris, Lens culinaris, Cicer arietinum, Glycine max, Pisum sativum, Medicago sativa) üç farklı strese (kuraklık, sıcaklık, tuz) maruz bırakılmıştır. Örneklerin biyokimyasal olarak prolin miktarları belirlenmiş ve sonrasında ortak primer kullanılarak gen ekpresyon seviyeleri hesaplanmıştır. Sonuçlarımızda tüm bitki örneklerinde prolin miktarları önemli ölçüde artmıştır. Gen ekspresyon analizleri ise bu sonuçlarla korelasyon göstermektedir. Bu çalışmada, stres koşullarında, bazı tarımsal öneme sahip baklagil bitkilerinin stres altında olduğunu gösteren, önemli bir belirteç olan prolin seviyelerini, gen ekspresyonu düzeyinde belirlemek adına kullanılabilecek ortak bir marker belirlenmiştir. Bulgularımız, prolin biyosentezinde görev alan genlerin ifadesini araştırılmasının, bitkilerin stres tepkilerini moleküler düzeyde anlamaya yardımcı olabildiğini göstermektedir. Ayrıca özellikle moleküler yöntemlerle yapılan analizler daha hızlı ve hassas sonuçlar verir. Bu, prolin biyosentezinde aktif olan genlerin dinamik değişimlerini daha kısa sürede gözlemlemeye ve gen düzeyindeki değişiklikleri, bitkinin strese karşı hazırlık aşamasını ya da erken yanıtlarını gözlemlemeye olanak sağlar.

Anahtar Kelimeler

• Leguminosae
• Abiyotik stres
• Prolin biyosentezi
• Pirolin-5-karboksilat sentetaz (P5CS)
• Gen ekspresyonu

Kaynakça

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