Physiological responses and P 5 CS gene expression of transgenic oil palm plantlet induced by drought stress

Kekeringan merupakan salah satu faktor pembatas dalam budidaya tanaman, seperti halnya pada kelapa sawit (Elaeis guineensis Jacq.). Pendekatan transgenik diharapkan mampu meningkatkan toleransi tanaman terhadap cekaman kekeringan dan meminimalisir rendahnya produktivitas saat terjadinya kekeringan. Prolin sebagai salah satu senyawa osmoprotektan pada tanaman yang biosintesisnya melibatkan gen P5CS dijadikan target rekayasa dalam penelitian ini. Penelitian ini bertujuan mengevaluasi tingkat ketahanan planlet kelapa sawit transgenik P5CS terhadap cekaman kekeringan menggunakan senyawa polietilena glikol 6000 (PEG-6000). Pada penelitian ini planlet kelapa sawit transgenik yang disisipi gen P5CS dan non-transgenik diperlakukan dengan PEG-6000 0, 2, dan 4% secara in vitro. Rancangan acak lengkap faktorial dengan tiga ulangan digunakan dalam penelitian ini. Skor tingkat kekeringan, kandungan klorofil total, kandungan karotenoid, kandungan prolin, dan ekspresi gen P5CS pada jaringan daun diamati pada 7 dan 14 hari setelah perlakuan cekaman. Hasil penelitian menunjukkan bahwa tanaman transgenik mempunyai skor tingkat kekeringan yang lebih rendah dibandingkan non-transgenik. Cekaman PEG-6000 pada konsentrasi 4% menurunkan kandungan klorofil total dan karotenoid yang lebih besar dibandingkan dengan konsentrasi 2% pada tanaman non-transgenik pada 7 dan 14 hari setelah perlakuan (HSP). Selain itu, tanaman transgenik mengalami peningkatan akumulasi prolin dan ekspresi gen P5CS selama perlakuan cekaman. Hasil ini menunjukkan bahwa transgen P5CS mampu meningkatkan toleransi tanaman kelapa sawit terhadap cekaman kekeringan.


Introduction
Oil palm (Elaeis guineensis Jacq.) is one of the important economic oil crops in the world. Oil palm has the highest yield per hectare of all oil crops, such as soy bean, rapeseed, and sunflower. Moreover, palm oil is the largest source of vegetable oil (Corley & Tinker, 2016). Indonesian Statistics (BPS, 2019) reported that during the year 2000-2018, the production of Indonesian oil palm increased 80.87%, while export activity of crude palm oil (CPO) increased 85.27%.
Drought stress tolerance in plants is one of the complex traits and controlled by many genes (Ashraf, 2010). Maintaining excess water loss during stress through osmoprotectants or compatible solutes known as a tolerance strategy to drought. Proline accumulation is an adaptive response and also known as an osmoregulatory solute in plants under hyperosmotic stresses, such as drought (Iskandar et al., 2014). The Δ 1pyrroline-5-carboxylate synthetase (P5CS) is a rate limiting enzyme that has a role in proline biosynthesis which is encoded by P5CS gene (Kishor et al., 2005). Proline also reported as a biochemical marker under drought stress in plants (Toruan-Mathius et al., 2004;Ashraf, 2010;Fichman et al., 2015;Zarattini & Forlani, 2017). In addition, drought stress has also been reported to interfere the photosynthetic system of plants as indicated by decreasing of chlorophyll content (Din et al., 2011;Jazayeri et al., 2015). Besides reducing chlorophyll, drought stress also reduces carotenoids due to the increase of reactive oxygen species production (Mibei et al., 2017). As common non-enzymatic antioxidant, carotenoids protect cells from excess damage during the stress (Ghobadi et al., 2013). Chorophyll and carotenoids also reported as useful traits for identification the drought tolerance level of plants (Talebi et al., 2013).
The availability of drought tolerant of oil palm plant material is needed. One of strategy that can be conducted to obtain drought tolerant of oil palm is via genetic engineering. Transformation and genetic engineering in plants using the P5CS gene has been carried out on various plants, i.e. tobacco (Riduan et al., 2010;Zarei et al., 2012), sugarcane (Minarsih et al., 2015), wheat (Pavei et al., 2016), and oil palm (Budiani et al., 2019). Furthermore, Budiani et al. (2019) successfully developed P5CS-transformed oil palm embryonic calli into plantlets but these plantlets have not been evaluated yet under certain stress conditions, especially to drought stress. In the present study, we evaluated the physiological responses and P5CS gene expression of transgenic oil palm plantlets under drought stress using PEG-6000. The findings could be useful and become a basis knowledge for improving oil palm productivity under related water deficit stress.

Experiment conditions
The experiment was conducted using Murashige-Skoog culture medium containing PEG-6000 as a drought treatment. There were three treatments in this study, i.e. 0% PEG-6000 (-0.24 MPa) as control, 2% PEG-6000 (-0.98 MPa), and 4% PEG-6000 (-2.52 MPa) as stress conditions according to Cha-um et al. (2010). These treatments were exposed to non-transgenic and P5CS transgenic plantlets with the criteria as described above. The experiment was arranged as a factorial completely randomized design with three replications (n = 3). Drought level score, total chlorophyll-, carotenoid-, proline content, and P5CS gene expression were measured in this study. These parameters were observed at 7 and 14 DAT.

Drought level score determination of oil palm under PEG-6000-induced stresses
The level of PEG-6000-induced drought was represented as a score of 0 (without drying area on the leaves) to 9 (the plant died) according to the leaf drying area at the vegetative stage (IRRI, 2002). The drought level score determination was done at 1, 7, and 14 DAT.

Total chlorophyll and carotenoid content determination
The total chlorophyll and carotenoid contents were extracted by acetone 80%. Briefly, 100 mg of fresh leaf from treated plants were ground in 10 mL of cold acetone 80% (v/v) for total chlorophyll and carotenoid pigments extraction. The extract was centrifuged at 3000g at 4 °C for 15 minutes (Turhadi et al., 2019). The supernatant was read at 470, 663, and 646 nm using Thermo Scientific TM Multiskan TM GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc., USA). The chlorophyll and carotenoid were determined according to a formula by Lichtenthaler (1987) at 7 and 14 DAT in three plantlets in each treatments.

Proline content determination
Proline content was determined according to Bates et al. (1973). Briefly, 0.25 g of fresh leaf were ground using 5 mL of sulfosalicylic acid 3% (w/v). The extracts were centrifuged at 10000 rpm at 25 °C for 10 minutes. About 2 mL of supernatant was mixed with 2 mL of acid-ninhydrin reagent (1.25 g ninhydrin in 30 mL of glacial acetic acid and 20 mL of 6 M phosporic acid) and 2 mL of glacial acetic acid. The mixture was then incubated at 100 °C for 1 hour and immediately soaked in ice water. The filtrate was extracted using 4 mL toluene and vortexed. The proline content of leaves which harvested on 7 and 14 DAT was determined at λ 520 nm using Thermo Scientific TM Multiskan TM GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc., USA).

RNA isolation and quantitative real timepolymerase chain reaction (qRT-PCR)
P5CS gene expression level was done at 7 and 14 DAT. Total RNA was isolated from leaf tissues of treated plants using TRIzol TM reagent (Invitrogen, USA) according to the manufacturer's protocols. RNAs were treated with the DNase I kit (Sigma-Aldrich ® , USA) to remove contaminating genomic DNA according to the manufacturer's protocols. The cDNAs were then synthesized using AccuPower ® CycleScript RT Premix (dT20) (BIONEER, USA) according to the manufacturer's protocols. The synthesized cDNAs were used for the P5CS gene expression level by qRT-PCR. The P5CS primers used in the expression analysis was designed based on P5CS gene sequence of Vigna aconitifolia with Genbank accession no. M92276.1. The qRT-PCR in each sample was performed in triplicates using genespecific primers (Table 1) and SensiFAST TM SYBR ® Hi-ROX (Bioline, USA) according to the manufacturer's protocols. The Actin primer used for this quantification as a reference gene ( Table  1). The expression level of the P5CS gene was quantified by 2 -∆∆Ct formula (Schmittgen & Livak, 2008).

Statistical analysis
Data were statistically analyzed using analysis of variance (ANOVA) (α=0.05) and correlation analysis (α=0.05). Analysis of variance was performed in SPSS 16.0 program (SPSS Inc., Chicago, IL, USA), while correlation analysis was performed and visualized in R-Studio program using corrplot package.

Drought level score of non-and transgenic oil palm under polyethylene glycol 6000-induced stresses
Non-and transgenic plantlets demonstrated different responses to drought stress treatment using PEG-6000 in this present study. Leaves of non-transgenic plantlets showed drought response in 4% PEG-6000 at 7 DAT. The increase of PEG-6000 concentration and longer duration of treatment caused an increase of drought symptom especially in non-transgenic oil palm plantlets as shown in Figure 2.
The drought level between transgenic and nontransgenic plantlet leaves showed clearly different at 14 DAT under 4% PEG-6000 treatment. The leaves of the transgenic plantlets showed mostly green (average score = 1.0), while non-transgenic plantlets showed brown color or dry (average score = 5.0) (Figure 3). Lower scores indicated low level of drought stress and vice versa. These results indicated that P5CS-transgenic oil palm had higher tolerance level to drought stress than nontransgenic.

Total chlorophyll, carotenoids, and proline content in transgenic oil palm
The response of P5CS-transgenic oil palm to drought stress conditions was demonstrated by measuring several physiological parameters, i.e. total chlorophyll-, carotenoids-, and proline content in leaf tissues (Figure 4a-d & 5). Total chlorophyll content significantly (p<0.05) decreased within interaction between genotypes and PEG-6000 concentration (Figure 4a). Transgenic plantlets showed higher total chlorophyll content than that of non-transgenic under 2 and 4% PEG-6000 (Figure 4a). Total chlorophyll content of non-transgenic plantlets decreased by 61% in compared to the control at 14 DAT ( Figure 4b). Conversely, the transgenic plantlets were able to maintain the total chlorophyll content even under PEG-6000 stresses. The total chlorophyll content in transgenic plantlets under 4% PEG-6000 showed an increase of 31% and 15% at 7 and 14 DAT, respectively. Based on these responses indicated that P5CS transgene from Vigna aconitifolia increased the oil palm tolerance level to drought stress.   Drought level score /

Skor tingkat kekeringan
Observation at-(Day After Treatment) / Pengamatan pada-(Hari Setelah Perlakuan) The decreasing of oil palm's chlorophyll content under PEG-6000 treatment using in-vitro conditions also reported in previous research studies (Cha-um et al., 2010(Cha-um et al., , 2012. Moreover, the decreasing of chlorophyll content under drought stress also reported in oil palm using watering intensity treatments (Cha-um et al., 2013;Azzeme et al., 2016). In this present study, the decreasing of total chlorophyll content under PEG-6000 treatment was suggested due to the damage of chloroplast organelle during stress.
Beside of total chlorophyll content, the increase of tolerance level of transgenic oil palm plantlets was also shown in carotenoids profile content. There was significantly (p<0.05) interaction between genotypes and PEG-6000 concentration to carotenoids content of oil palm plantlets. The carotenoids content of transgenic plantlets under 4% PEG-6000 higher than that control and 2% PEG-6000 (Figure 4c). Our result showed that carotenoids content of transgenic plantlets under 4% PEG-6000 increased by 16% and 26% at 7 and 14 DAT, respectively. Conversely, the carotenoids content of nontransgenic plantlets under 4% PEG-6000 decreased by 47% and 37% at 7 and 14 DAT, respectively (Figure 4d).
The increase of carotenoids content in transgenic plantlets suggested of having a role in the tolerance mechanism of oil palm to drought stress. Besides as light-harvesting pigment, carotenoids also have a role as chlorophyll protecting pigment and protect the chloroplast from photooxidative damage as well (Wang et al., 2014;Das & Roychoudhury, 2014). Moreover, drought is an abiotic stress that produces reactive oxygen species (ROS). According to Das & Roychoudhury (2014) carotenoids as pigment that produced in chloroplast and other organelles (nongreen plastids) involved in the non-enzymatic detoxification of ROS.
Oil palm transgenic significantly (p<0.05) had higher proline content than that non-transgenic plantlets (Figure 5a). In addition, the proline content also increased during the stress especially, in the transgenic plantlets. A higher increase of proline content was shown by transgenic compared to non-transgenic plantlets (Figure 5b). The proline content of transgenic under 4% PEG-6000 at 14 DAT increased by 273%, while in nontransgenic plantlets increased by 102%.
The presence of P5CS transgene that controlled by 35S-CaMV promoter caused the increase of proline content in oil palm transgenic plantlets during stress. Toruan-Mathius et al. (2004) stated that proline content is one of the biochemical marker of drought stress in oil palm. In our present study, the proline content in transgenic plantlets sharply increased during the stress. Borgo et al. (2015) stated that proline accumulation is related to the tolerance level of Vigna aconitifolia under drought stress conditions. Proline is suggested to be involved in the chloroplast protection by quenching reactive oxygen species in Arabidopsis thaliana (Moustakas et al., 2011).
Various studies found that proline content also increased after treated with drought stress. The P5CS-transgenic tobacco showed increase of proline content compared non-transgenic ranged 3618 -4449 and 2044 µg/g fresh weight, respectively (Riduan et al., 2010). The increase of proline content in those transgenic plants improved the tolerance level to drought stress.

P5CS gene expression in transgenic oil palm increased during stresses
The increase of proline content was in line with the increase of P5CS gene expression. The increase of P5CS gene expression in transgenic plantlets under 2 and 4% PEG-6000 was higher than that of in non-transgenic plantlets ( Figure 6). This result supported the hypothesis about the tolerance level of transgenic oil palm related to the increase of proline accumulation. According to Fichman et al. (2015) Δ 1 -pyrroline-5-carboxylate synthetase which is encoded by P5CS is one of the key enzyme in proline biosynthesis. The increase of proline content also reported that no toxic effect to mitochondria and chloroplast ultrastructure of VaP5CS129A transgenic plants under 12 days of water deficit conditions (Borgo et al., 2015).

Relationship between physiological and P5CS gene expression level in oil palm during PEG-6000 stress treatment
Carotenoids significantly (p<0.05) and positively correlated with chlorophyll content at 7 and 14 DAT. In addition, the P5CS gene expression at 7 and 14 DAT also showed positive correlation with proline content (Figure 7). Bandurska et al. (2017) also reported positive correlation between proline content and activity of P5CS protein in barley (Hordeum vulgare L.) under drought stress. Our present study showed a significantly negative correlation (p<0.05) between leaf drought level score and chlorophyll content (Figure 7). As a consequence of drought level increased, the chlorophyll content of oil palm in leaves tissues decreased.

Conclusion
Drought stress induced by PEG-6000 resulted physiological changes in oil palm transgenic plantlets showing improved drought tolerance potential. The 4% PEG-6000 clearly showed different response between P5CS-transgenic and non-transgenic oil palm plantlets on their leaves drought level score. The drought stress during 14 days treatment significantly decreased chlorophyll and carotenoids content in non-transgenic plantlets, while in transgenic planlets. The P5CStransgene increased the tolerance level of oil palm under PEG-6000 stress as shown by the increasing of proline accumulation and P5CS gene expression in leaf tissues.