Some Biological Aspects and Proline Metabolism Genes as Influenced by Polyethylene Glycol and Salicylic Aacid in Two Wheat Cultivars

The aim of this work was to study the response of two wheat cultivars with different drought tolerance to PEG (-0.4 Mpa) and SA (0.5 mM). Data of seed germination and seedling growth indicated that Misr-2 cultivar was more drought tolerant than Gemmeiza-12. Under non- stress conditions, Misr-2 had higher values of pigments, soluble sugars (Glu, Fru and Suc) and activities of antioxidant enzymes (CAT, APOX, GPOX and SOD) but lower values of proline than those of Gemmeiza-12. The cultivar with a higher proline content (Gemmeiza-12) compared to the other cultivar had a higher expression of proline synthesizing gene (P5CS) but lower expression of proline degrading genes (PDH and P5CDH). Under drought conditions, the drought-sensitive (Gemmeiza-12) needed more osmoregulators and antioxidants than drought-tolerant cultivar (Misr-2) to minimize the negative effects of water and associated oxidative stresses. Therefore, soluble sugars, proline content and anti-oxidation enzymes were higher in Gemmeiza-12 than Misr-2. Also, under drought stress, up-regulation of P5CS and down-regulation of PDH and P5CDH in Gemmeiza-12 were higher than Misr-2. While treatment of the stressed plants with SA up-regulated P5CS gene in both cultivars, down-regulation of PDH and P5CDH in Gemmeiza-12 was clearer than Misr-2.

Functional Characterization of the Stipa purpurea P5CS Gene under Drought Stress Conditions

Free proline has multiple functions in plant cells, such as regulating osmotic potential and protecting both proteins and cell membranes. The expression of Δ1-Pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway, increases under drought, salt and cold stress conditions, causing plant cells to accumulate large amounts of proline. In this study, we cloned and identified the P5CS gene from Stipa purpurea, which has a full-length of 2196 bp and encodes 731 amino acids. A subcellular localization analysis indicated that SpP5CS localized to the cytoplasm. The ectopic overexpression of SpP5CS in Arabidopsis thaliana resulted in higher proline contents, longer roots, higher survival rates and less membrane damage under drought stress conditions compared with wild-type controls. SpP5CS-overexpressing A. thaliana was more resistant to drought stress than the wild type, whereas the deletion mutant sp5cs was less resistant to drought stress. Thus, SpP5CS may be a potential candidate target gene for increasing plant resistance to drought stress.

Physiological responses and P5CS gene expression of transgenic oil palm plantlet induced by drought stress

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.

Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium-mediated transformation

AbstrakCekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]Abstract Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS (Δ1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]

Proline as a probable biomarker of cold stress tolerance in Sorghum (Sorghum bicolor)

Plants have developed physiological and molecular mechanisms to support and adapt to adverse environments. One response to abiotic stress is the accumulation of free proline (PRO). PRO can induce the expression of many genes, which have the proline-responsive element (PRE) in their promoters, nevertheless due to the complexity of interactions between stress factors and various molecular, biochemical and physiological phenomena it is still unclear whether a more efficient PRO accumulation can be considered a biomarker of tolerance in plants. In the present work, we evaluated the accumulation of PRO in two genotypes of sorghum with contrasting tolerance to cold stress. To explore the cause behind the accumulation of proline under cold stress conditions, we identified the Transcription Factors Binding Sites (TFBS) present in the promoter regions in the genes involved in the biosynthesis and degradation of proline in sorghum and other important crops, finding that the untranslated 3 'region P5CS gene contains different TFBS. We found TFBS that could allow the activation of genes involved in proline biosynthesis through the ornithine pathway under cold stress conditions, suggesting that ornithine route can be activated under cold stress conditions

Over Expression of p5cs Gene to Increase Cold Stress Tolerance in Iranian Species of Petunia

In order to examine the relationship between proline and cold stress in Iranian (local) genotype of petunia, p5cs gene was transferred to petunia through Agrobacterium-mediated transformation. The leaf discs from four weeks and shoot apices from 7-day-old petunia in vitro plants were co-cultivated with Agrobacterium strain LBA4404 harboring a plasmid pBI121 as the vector system for transformation of petunia. pBI121 plasmid containing β-glucuronidase (gus) gene as a reporter gene and Δ1-pyrroline-5-carboxylate synthetase (p5cs) gene and neomycin phosphotransferase (nptII) gene were used as a selectable marker. The co-cultivated leaf discs and shoot apices were transferred to the selective medium and thereafter to proliferation medium respectively. To confirm transformation, regenerated plants were subjected to the polymerase chain reaction (PCR), GUS histochemical and proline assays. The results confirmed the presence of the gus and p5cs gene in the genome of all transformants and the transformed plants were more tolerant (674.87 μg/g fresh leaves) than the wild types under stress conditions.Plant Tissue Cult. & Biotech. 28(1): 35-44, 2018 (June)

Keefektifan Agrobacterium mentransfer gen P5CS ke dalam kalus tebu klon PS 851 Effectiveness of Agrobacterium to transfer P5CS gene into sugarcane callus PS 851 clone

Summary Transformation of a P5CS gene construct into plant cells coupled with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The purpose of the research is to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus. In this method, recombinant plasmid of pBI-P5CS could be transferred into host cells of Agrobacterium LBA4404 through triparental mating with pRK2013 helper. The parameters were tested to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus were the addition of antioxidant and 1.0% glucose, callus age (2, 3, and 4 weeks), medium pH (4.5; 5.0; and 5.6), treated with air dry for 30 minutes, wetting agent of silwet with and without short vacuum treatment, and acetosyringone consentration (100, 500, and 1000 mg/L). Identification of the transgene in sugarcane was conducted by PCR using spesific primers, and the expression was tested by measuring of the proline content. The result showed that addition of acetosyringone 100 ppm or more, P5CS transfer into the sugarcane explants by Agrobacterium was effective. The genetic transformation could be optimized by selecting proper age of calli, which was four weeks after sub-culture. The effectiveness could be maintained and slightly improved by inoculation at pH 4.5, addition 1.0% glucose, wetting agent of silwet with short vacuum treatment, or treated with air drying for 30 minutes. In vitro cultures for transgenic regeneration required addition of antioxidant to prevent browning in the culture media. The amplified DNA fragment demonstrated that the gene was transferred into sugarcane plantlets, and P5CS gene expression showed increasing proline content in transgenic sugarcane plantlets.Ringkasan Transformasi transgen P5CS yang diikuti dengan regenerasi tanaman transgeniknya diper-kirakan mampu menghasilkan tanaman tebu transgenik yang toleran terhadap cekaman kekeringan. Penelitian ini bertujuan untuk me-ningkatkan efektivitas dan efisiensi Agro-bacterium mentransfer gen P5CS ke dalam kalus tebu. Dalam metode ini, plasmid rekombinan pBI-P5CS berhasil dengan baik ditransformasi-kan ke dalam sel. Agrobacterium LBA4404 dengan pendekatan triparental mating meng-gunakan helper pRK2013. Parameter yang diuji untuk meningkatkan kondisi efektif dan efisien dalam transfer gen P5CSke dalam kalus tebu adalah penambahan antioksidan dan glukosa 1,0%, umur kalus (2, 3, dan 4 minggu), pH medium (4,5; 5,0; dan 5,6), pengeringan kalus 30 menit, bahan pembasah silwet tanpa dan dengan pemakuman, dan konsentrasi aseto-siringon (100, 500, dan 1000 mg/L). Pengujian keberadaan transgen P5CS dilakukan dengan PCR menggunakan primer spesifik, sedangkan ekspresinya diuji dengan mengukur kandungan prolin dari tanaman tebu. Hasil percobaan menunjukkan bahwa dengan penambahan asetosiringon 100 ppm atau lebih, penggunaan Agrobacterium terbukti efektif dan efisien dalam transfer konstruk transgen P5CS ke dalam eksplan kalus tebu. Transformasi dapat dioptimalkan dengan memilih eksplan kalus tebu yang baik, yaitu yang umur subkulturnya empat minggu. Efektivitasnya juga dapat dijaga atau sedikit ditingkatkan dengan inokulasi pH 4,5, penambahan glukosa 1,0%, bahan pembasah silwet dengan pemakuman, ataupun pemberian perlakuan pengeringan udara selama 30 menit. Kultur kalus transgenik memerlukan penambahan antioksidan untuk mencegah terjadinya pen-cokelatan. Adanya fragmen DNA hasil amplifikasi dengan primer spesifik P5CS menunjukkan pada tanaman tebu telah terdapat gen P5CS. Demikian pula dengan ekspresi gen P5CS, menunjukkan adanya peningkatan kandungan prolin pada tanaman tebu transgenik.

Keefektifan Agrobacterium mentransfer gen P5CS ke dalam kalus tebu klon PS 851 Effectiveness of Agrobacterium to transfer P5CS gene into sugarcane callus PS 851 clone

Summary Transformation of a P5CS gene construct into plant cells coupled with regeneration for transgenic plantlets should develop sugarcane tolerant to drought stress. The purpose of the research is to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus. In this method, recombinant plasmid of pBI-P5CS could be transferred into host cells of Agrobacterium LBA4404 through triparental mating with pRK2013 helper. The parameters were tested to increase the effectiveness and efficiency of Agrobacterium transferring the gene into sugarcane callus were the addition of antioxidant and 1.0% glucose, callus age (2, 3, and 4 weeks), medium pH (4.5; 5.0; and 5.6), treated with air dry for 30 minutes, wetting agent of silwet with and without short vacuum treatment, and acetosyringone consentration (100, 500, and 1000 mg/L). Identification of the transgene in sugarcane was conducted by PCR using spesific primers, and the expression was tested by measuring of the proline content. The result showed that addition of acetosyringone 100 ppm or more, P5CS transfer into the sugarcane explants by Agrobacterium was effective. The genetic transformation could be optimized by selecting proper age of calli, which was four weeks after sub-culture. The effectiveness could be maintained and slightly improved by inoculation at pH 4.5, addition 1.0% glucose, wetting agent of silwet with short vacuum treatment, or treated with air drying for 30 minutes. In vitro cultures for transgenic regeneration required addition of antioxidant to prevent browning in the culture media. The amplified DNA fragment demonstrated that the gene was transferred into sugarcane plantlets, and P5CS gene expression showed increasing proline content in transgenic sugarcane plantlets.Ringkasan Transformasi transgen P5CS yang diikuti dengan regenerasi tanaman transgeniknya diper-kirakan mampu menghasilkan tanaman tebu transgenik yang toleran terhadap cekaman kekeringan. Penelitian ini bertujuan untuk me-ningkatkan efektivitas dan efisiensi Agro-bacterium mentransfer gen P5CS ke dalam kalus tebu. Dalam metode ini, plasmid rekombinan pBI-P5CS berhasil dengan baik ditransformasi-kan ke dalam sel. Agrobacterium LBA4404 dengan pendekatan triparental mating meng-gunakan helper pRK2013. Parameter yang diuji untuk meningkatkan kondisi efektif dan efisien dalam transfer gen P5CSke dalam kalus tebu adalah penambahan antioksidan dan glukosa 1,0%, umur kalus (2, 3, dan 4 minggu), pH medium (4,5; 5,0; dan 5,6), pengeringan kalus 30 menit, bahan pembasah silwet tanpa dan dengan pemakuman, dan konsentrasi aseto-siringon (100, 500, dan 1000 mg/L). Pengujian keberadaan transgen P5CS dilakukan dengan PCR menggunakan primer spesifik, sedangkan ekspresinya diuji dengan mengukur kandungan prolin dari tanaman tebu. Hasil percobaan menunjukkan bahwa dengan penambahan asetosiringon 100 ppm atau lebih, penggunaan Agrobacterium terbukti efektif dan efisien dalam transfer konstruk transgen P5CS ke dalam eksplan kalus tebu. Transformasi dapat dioptimalkan dengan memilih eksplan kalus tebu yang baik, yaitu yang umur subkulturnya empat minggu. Efektivitasnya juga dapat dijaga atau sedikit ditingkatkan dengan inokulasi pH 4,5, penambahan glukosa 1,0%, bahan pembasah silwet dengan pemakuman, ataupun pemberian perlakuan pengeringan udara selama 30 menit. Kultur kalus transgenik memerlukan penambahan antioksidan untuk mencegah terjadinya pen-cokelatan. Adanya fragmen DNA hasil amplifikasi dengan primer spesifik P5CS menunjukkan pada tanaman tebu telah terdapat gen P5CS. Demikian pula dengan ekspresi gen P5CS, menunjukkan adanya peningkatan kandungan prolin pada tanaman tebu transgenik.