BIOTROPIA Vol. 28 No. 3,2021: 181 - 192 DOI: 10.1 1598/btb.2021.28.3.1047 OVEREXPRESSION OF Ga! GENE INCREASES GROWTH AND HYPOSALINE TOLERANCE IN Kappaphycus aalvarezii TRANSGENIC PLANTLETS ERINA SULISTIANI1*, SUHARSON02, ENCE DARMO JAYA SUPENA2 AND MIFTAHUDIN2 'SEAniZEO BIOTROP, Jalan Rdya TdurKm 6, Bogor 16134, Indonesia 'Department of Biolo~, Faculiy ofniathematics and Natural Sciences, Institzit Pertanian Bogor, Bogor 16680, Indonesia. Received 4 April 2018/Accepted 27 June 2020 ABSTRACT G proteins are membrane proteins that play roles in signal transduction in living organisms. They consist of a, p and y subunits. The G protein a subunit (Ga) plays a role in plant resistance toward biotic and abiotic environmental stresses. Transgenic plantlets of Kappaph_yczis aluare+i carrying the Ga gene (derived from soybean) have been successfully obtained through Agrobacterium tnmefaaciens-mediated transformation. The present study aimed to: 1. compare the growth of non-transgenic and transgenic plantlets of K. alvareei in vitro using Provasoli enriched seawater (PES) medium with normal salinity and hyposalinity and 2. analyze the expression level of the Ga gene in transgenic plantlets using quantitative Polymerase Chain Reaction (qPCR). The results showed that all transgenic plantlets (six clones) had significantly higher daily growth rate (DGR, %/d) than that of non- transgenic under the condition of normal salinity (30 ppt) and hyposalinity (15 and 20 ppt) for 5 weeks of observation. At 15 ppt, transgenic plantlets were more tolerant than non-transgenic ones, as most thalli of transgenic plantlets remained brown in color, whereas most thalli of non-transgenic plantlets were bleached. The results of the qPCR analysis showed that the expression of the Ga gene in transgenic plantlets increased by 6.43 - 8.03 times compared with that of non-transgenic plantlets. The result of Pearson correlation analysis showed that relative expression of Ga gene had a strong correlation, both with DGRs in normal salinity and hyposalinity of transgenic plantlets (correlation coefficient > 0.7). The correlation was linearly positive, where increased expression of the Ga gene was strongly associated with an increase in DGRs. Keywords: Ga gene overexpression, hyposaline tolerance, K@aph_ycus alvareei, transgenic plantlet INTRODUCTION Kappapbctls alvareei Doty is a commercially important species of red algae (Rhodophyta) that is known in the trade as 'Cottonii seaweed'. This species is the main source of kappa (x)- carrageenan, which is widely used in the pharmaceutical, food and cosmetics industries. K alvare?? is widely cultivated in coastal waters of Indonesia and Philippines. However, in the cultivation of K. alvareei in coastal waters, major problems may occur such as salinity change and other frequent pronounced fluctuations in environmental factors in coastal waters. K al'vare?? grows at a salinity of 28 - 34 ppt (part *Corresponding author, email: esulistiani@biotrop.org per thousand) (Parenreng et al. 201 1). However, in rainy season with heavy rainfall, the salinity of seawater can decrease up to below 28 ppt. The decrease of salinity subjects the seaweed to hyposaline stress. The stress can cause the occurrence ice-ice dsease in seaweed, which symptoms include slow growth, bleaching and decay of some or all of the thalli (Parenrengi e t al. 2011). To overcome the impacts of ice-ice disease in seaweed, a genetic transformation needs to be done to obtain K ahare?? that is tolerant to hyposaline stress. The heterotrimeric G proteins are membrane proteins consisted of the a, p and y subunits (Ga, GP and Gy) (Urano e t al. 201 3). They play a role in signal transduction in a living organism as mediators that send extracellular signals from BIOTROPIA Vol. 28 No. 3,2023 receptor molecules in the cell membrane to MATERIALS AND METHODS effector molecules. In plants, Gcc proteins play a role on cell growth, cell proliferation, defense against hsease, stomata1 movement, channel regulation, sugar sensing and some hormonal responses (Urano et al. 2013). Moreover, according to Chakraborty et al. (2015), Ga proteins play a role in plant resistance to biotic and abiotic environmental stresses. Seaweeds respond to changes in salinity by adjusting the balance of osmotic potential between internal and external cells via pumping of inorganic ions such as I<+, Na+ and C1- into or out of the cells (Karsten 2012). The influx of external Ca2+ into the cytoplasm facilitates the maintenance of I<+ and Na+ homeostasis during salinity stress because Ca2+ plays a role in activating the Na+/Hf Salt-Overly-Sensitive1 (SOS1) antiporter and the cation/Ht NHX antiporter (Hasegawa 201 3). In hyposaline condition, Ca2+ also plays a role in reducing the permeability of the plasma membrane to other ions, thereby reducing ion loss that occurs during water influx (Hurd et al. 2014). In addition, Ca2+ plays a role in the regulation of aquaporin, which is a water channel protein (Gilliham et al. 201 1) recently found in algal cell plasma membranes as well (Anderberg et al. 201 1). Gcc protein signaling plays a role in Ca2+ influx to the cytoplasm, owing to the abhty of Ga protein in regulating Ca2+ channels in plasma membranes (Gelli et al. 1997; Aharon et al. 1998; Thuleau et al. 1 998; Zhang et a/. 201 1). Based on the above description, overexpression of the Ga gene in K alvareei is expected to increase Ca2+ influx to the cytoplasm and increase the tolerance of ths seaweed to hyposaline stress (< 28 ppt). Transgenic plantlets of K. alvareei carrying the Ga gene (derived from soybean) have been - successfully obtained through Agrobacte~am tamefdciens-mediated transformation (Sulistiani et al. 2019). The study aimed to: 1. conduct growth performance test of non-transgenic and transgenic plantlets of K. alvare~i in an in vitro culture medium with normal salinity and hyposalinity and 2. analyze the expression level of the Ga gene in non-transgenic and transgenic plantlets using qPCR method. Growth Performance Test of Non- transgenic Plantlets in Hyposaline Condition Non-transgenic plantlets were regenerated from callus which induced from apical thallus of K alvare@i (collected from Takalar, South Sulawesi, Indonesia). The callus was grown into plantlets by culturing the plantlets in Provasoli Enriched Seawater (PES) medium (Provasoli 1968) for 6 months (Sulistiani & Yani 2014). The experiment was conducted to observe the growth of non-transgenic plantlets in PES medium with normal salinity and hyposalinity. The plantlets (about 3 g) were grown in an aerated culture for 12 weeks with five treatments, i.e., PES with 5 different salinity levels: 15, 20, 25, 30, and 35 ppt (1,000 mL/bottle). Each treatment was conducted in three replicates. The cultures were continuously aerated with an air blower and placed in a culture room at temperature of 22 - 24 "C, lighting of 1,500 lux and a 12 : 12-h light and dark cycle. The medium was replenished weekly. The natural seawater used in this study had a salinity level of 32 ppt. The lower-than-32-ppt salinity level was prepared by adding distilled water to the natural seawater and stirring the solution using a magnetic stirrer until the desired s a h t y level was reached. In contrast, to obtain salinity level of more than 32 ppt, NaCl was added to the natural seawater and stirred unul reaching the desired salhty level. Salinity was measured using a refractometer. The plantlets were weighed weekly, and the daily growth rates (DGRs, %/d) were calculated using the formula of Dawes et al. (1 994): Ln (final weight) - Ln (initial weight) DGR = .o. o f d q x 100 Seawater with salinity level causing significant stress and decreased DGRs in the non- transgenic plantlets was used in the growth performance tests of transgenic plantlets in a hyposaline medium to select transgenic plantlets with tolerance to hyposalinity. Overexpression of Gcl gene in Kappapbycu~ aluareei transgenic plantlets - Sulistiani et al. Tolerance Test of Transgenic Plantlets in Hyposaline Condition An experiment was conducted to determine the tolerance of transgenic plantlets in PES medium with hyposaline condition, which exerted stress on the non-transgenic plantlets. Six clones of transgenic plantlets (Ll, E2, E5, E6, E7 and E8) were selected from a previous study. PCR analysis was used to confirm that the transgenic plantlets carried the Ga gene with a constitutive promoter CaMV 35s (Sulistiani et al. 2019). Transgenic plantlets were regenerated from genetically transformed callus using the same method as that used for the non- transgenic plantlets. The transgenic plantlets (about 0.3 g) were grown in 500 mL of PES mehum with three hfferent salinity: 30 ppt (normal salinity), 20 ppt and 15 ppt (hyposalinity). For comparison (control), non-transgenic plantlets were also grown in the same salinity treatments. The experiment was conducted for 5 weeks, with each salinity treatment being performed in three replicates. The plantlets were weighed weekly and the DGRs were calculated. One sample of each of the non-transgenic and transgenic plantlets (each about 4 g) was taken for calcium content analysis. The method of analysis was based on SNI 2354.5: 2011 (BSN- National Standardization Agency of Indonesia 201 I), and the calcium content was measured using an Atomic Absorption Spectrophotometer (AAS). Quantification of GLY Gene Expression in Transgenic Plantlets using qPCR Total RNA was extracted from two clones of putative transgenic plantlets and one clone of non-transgenic plantlets of K. alvare?? (6 months old in an aerated culture) using TRIzolB reagent (Invitrogen), and cDNA was synthesised using the IscriptTM cDNA synthesis h t (Bio-Rad). Extractions of total RNA and synthesis of cDNA were performed with three biological replicates for each clone. Quantification of Ga gene expression in transgenic and non- transgenic of K ahare?? was performed using Quantitative Polymerase Chain Reaction (qPCR) Applied Biosystems StepOnePlusm Real-Time PCR System. The Ga cDNA was amplified using the specific primer GA-F 5'- CAC GAG GTT GCT TTC TAG TTT C -3 'and GA-R 5'- ATG AGC TTC ACG AAA ACG AC -3'. The actin gene of K. ahare@' was used as an internal control of gene expression with the following specific primers: ICAct-F: 5'-CCG TCC CGA TIT ACG AGG GTTA-3' and K-Act-R: 5'- GCA TGA GGA GCT TCG CCA TCC-3' (Rajamuddin 2016). The Ga cDNA and actin cDNA were amplified in two replicates for each biological replicate. qPCR for Ga cDNA and actin cDNA was performed in a 10 pL reaction volume containing 200 ng cDNA, 2.5 pmol forward primer, 2.5 pmol reverse primer, 5 pL of S Y B R ~ ~ Select Master M x and nuclease-free water. The cDNA Ga gene was amplified as follows: pre-denaturation at 95 "C for 3 min, followed by 40 cycles of denaturation at 94 "C for 10 s, and annealing at 57 "C for 40 s. The amplification of actin cDNA was performed using the same protocol as that followed for Ga cDNA, except that annealing was performed at 55 "C. Quantification of the expression level was based on the expression of Ga cDNA relative to that of the reference gene (actin gene of K. ahare@) using the 2-MCt method. The calculation was performed by comparing the value of Ct (cycle threshold) from the expression of the two genes. Relative gene expression was represented by 2"Act, where M C t = Act transgenic - Act non-transgenic and Act = Ct target gene - Ct reference gene (Livak & Schmittgen 2001). Statistical Analysis Statistical analyses of DGR data were determined using analysis of variance (ANOVA) and Duncan's Multiple Range Test. The statistical analyses were performed in IBM SPSS Statistics 19.0 for Windows. RESULTS AND DISCUSSION Effect of Salinity on Growth and Development of Non-transgenic Plantlets The results of statistical analysis showed that the salinity of the culture mehum significantly affected the DGRs of K. ahare?? plantlets. The highest DGRs upon cultured for 12 weeks in PES mehum were obtained at s a h t y of 25 - 30 ppt (1.9 - 2.4O/o/d; Fig. 1). Based on the BIOTROPIA Vol. 28 No. 3,2021 literature, the seaweed K. alvareei grows well in coastal waters with a salinity of 28 - 34 ppt (Parenrengi e t al. 201 1). The results of this study showed that K. ahare?? plantlets cultured in PES medium with a salinity of 25 ppt still grew optimally, showing no significant difference from the DGRs of plantlets at a salinity of 30 ppt. At a salinity of 20 ppt, the plantlets still survived, but their DGRs were lower than DGRs of plantlets at a salinity of 30 ppt. This suggests that K. ahare?? plantlets that were regenerated from callus through somatic embryogenesis were tolerant to hyposalinity in vitro up to 20 ppt salinity. In hyposaline condtion, the Na' concentration outside seaweed cells is lower than that inside the cells. Therefore, water flows enter the cells following the osmotic gradient, whch in turn increases the volume of cells and their turgor. Under these conditions, the cells perform an osmotic adjustment mechanism by pumping inorganic ions such as K+, Na+ and C1- in and out from the apoplast to cytoplasm until the conditions return to the normal turgor pressure and homeostasis of I<+/Na+ is achieved (Hurd e t al. 2014). The decrease of DGRs upon exposure to hyposaline stress is due to the seaweed cells allocating most of their metabolic energy to the osmotic adjustment process, which in turn reduces the amount of metabolic energy used for growth (Karsten 2012). The growth decline is also due to the reduction of Oxygen Species (ROS) during hyposaline stress (Liu e t al. 2012). At 15 ppt sah ty , DGRs of K. ahare@ plantlet were sipficantly lower than those at 20 - 35 ppt salinity levels (Fig. 1). The weekly data indicated that the DGRs of plantlets cultivated in 15 ppt salinity level decreased during the first week of experiment (Fig. 2). At that time, the most of thallus plantlets changed color from reddish brown to white (bleaching), whch indcated that most of the thallus cells had died. The lowest DGR occurred in the third week (-O.lO/o/d), in which some of the bleached thalli began to soften and brittle, which reduced the plantlet weight compared to that at the beginning of the experiment. Hayashl et al. (2010) also cultured pieces of R alvareqi thallus originating from coastal waters in culture media with 15 ppt salinity level, the thallus experienced bleaching and ded withn three days. The osmotic adjustment mechanism was unsuccessful with extreme hyposaline stress (15 ppt), resulting in damage to the membranes, organelles and enzymes and ultimately seaweed cell death (Hurd et a/ 2014). In contrast, at 20 ppt salinity level, the decrease in the DGRs was not as severe as that at 15 ppt salinity level, because the thalli did not undergo bleaching. These results suggested that non-transgenic plantlets of K. ahare?? were not resistant or photosynthesis efficiency and photosynthetic sensitive to hyposaline stress at 15 ppt salinity fixation caused by the formation of Reactive level. Figure 1 Daily growth rates (DGRs) of KappapLyczls alvare+' non-transgenic plantlets for 12 weeks in PES medium with 15 - 35 ppt salinity Note: Numbers followed by the same letter are not significantly different, based on Duncan's multiple-range test at P < 0.01, n = 3. Overexpression of GE gene in Kz$$apbyccz/s alvamei transgenic plantlets - Sulistiani et al. 15 ppt m20 ppt Weeks Figure 2 Daily growth rates @GRs) of non-transgenic plantlets in PES medium with at 15 and 20 ppt salinity levels for 1 - 5 weeks of rearing At 15 ppt, a small part of the thalli of non- transgenic plantlets was still brown, especially at its base and tip (Fig. 3A). The brown part of the thallus grew thicker, which caused the DGRs to increase again at the fourth week (Fig. 2). At the 7h week, in the brown part of the thallus, many buds grew, but thallus branches did not grow until the 12& week of maintenance at 15 ppt salinity, with the thallus buds remaining short (Fig. 3B). When the plantlets were subcultured in PES medium with normal salinity (30 ppt), the thallus buds began to form thallus branches, whereas the bleached part of the thallus did not grow (Fig. 3C). After 2 months, the regenerated part of thallus began to separate itself from the bleached thallus and grew normally. At a salinity of 20 - 35 ppt, all of the plantlet thalli were still reddish brown. Under hyposaline condtion (20 ppt) (Fig. 4A), the plantlets formed more thallus branches than those under normal salinity (25 and 30 ppt) (Figs. 4B and 4C), but the thallus branches were short. In contrast, in hypersaline conditions (35 ppt), plantlets formed fewer thallus branches than those under normal salinity (Fig. 4D), and the thallus plantlets grow elongated. The results of this study indicated that the condition of the thallus branches can be used as an indicator of whether K. ahare?? grows in coastal waters with appropriate salinity seawater, hyposalinity or hypersalinity. Development of K. alvanei non-transgenic plantlets in PES medium with 15 ppt salinity: (A) most part of the thallus was bleached at the second week; (B) Growth of thdus buds at the brown part of the thallus at the seventh weeks; (C) Growth of thdus branches after plantlets had been subcultured in PES medium with normal salinity (30 ppt) Note: Scale bars = 2 mm. BIOTROPIA Vol. 28 No. 3,2021 Figure 4 Comparison of thallus branches of non-transgenic plantlets after 12 weeks in PES medium with different salinity: (A) 20 ppt; (B) 25 ppt; (C) 30 ppt; and (D) 35 ppt Note: Scale bars = 10 mm. Tolerance of Transgenic Plantlets in Hyposaline Conditions Based on the experimental results obtained in non-transgenic plantlets, six transgenic plantlets of K alva~eei (L7, E2, E3, E5, E6, E7 and E8) and one nontransgenic (N) plantlet were grown on PES medium only with a salinity of 15, 20 and 30 ppt for 5 weeks to test its tolerance to hyposaline stress. The result showed that at normal salinity, all of the transgenic plantlets had a significantly higher DGR (2.1 - 3.4% / d) than that of non-transgenic ones (1.4% / d) (Fig. 5). At 20 ppt salinity, all transgenic plantlets had higher DGRs (1.7 - 2.3% / d) than that of non-transgenic ones under normal salinity (1.4% / d) pig. 5), whereas non-transgenic plantlets had lower DGRs (0.8% / d) than that of non-transgenic ones in normal salinity. At 20 ppt salinity, non-transgenic and transgenic plantlets still survived, the color of the thalli was still brown (Fig. 6), but in the non- transgenic, most of the thallus brown color has faded (Fig. 6A). This shows that the tolerance of transgenic plantlets to 20 ppt salinity was higher than non-transgenic plantlets, because they can still grow faster than normal in these salinity, whereas the growth of non-transgenic plantlets was stunted or lower than the growth in normal salinity conditions. These results showed that overexpression of the Ga gene increased the DGRs of transgenic plantlets under the condition of normal salinity and 20 ppt. This is because the Ga protein plays a role in regulating cell proliferation and promoting growth as had been studied in Arabid0psi.r plants (Ullah 2001; Chen e t aL 2006; Colaneri et ak. 2014) and rice (Izawa 2010). Figure 5 Daily growth rates (DGRs) of non-transgenic (N) and transgenic (I. and E) plantlets under normal salinity (30 ppt) and hyposalinity (15 and 20 ppt) for 5 weeks observation. Note: Columns followed by the same letter are not significantly different based on Duncan's multiple-range test at p <0.01, n = 3). Overexpression of GCL gene in IGppaph_ycz/s alvareei transgenic plantlets - Sulistiani et al. Figure 6 Development of non-transgenic and transgenic thalli after 5 weeks at 20 ppt salinity. (A) non-transgenic; (B) transgenic Note: Scale bars = 5 rnm At 15 ppt salinity, all transgenic plantlets showed higher DGRs (0.8 - 1.2% / d) than those of non-transgenic plantlets (0.0% / d) (Fig. 5). In the first week, DGRs of non- transgenic plantlets decreased into -0.8% / d at 15 ppt salinity (Fig. 7). Most parts of the non- transgenic plantlet thalli (86% / d) was bleached after 3 days in PES medium with 15 ppt salinity (Fig. 8A), this caused the DGR of non- transgenic plantlets to fall below 0% / d during the first 2 weeks. Whereas, most parts of the transgenic plantlet thalli were still brown, with only 3 - 7% of the th& being bleached, until the 5& week of the experiment at 15 ppt salinity (Fig. 8B). Therefore, all of the transgenic plantlets still grew even with a lower DGRs (0.8 - 1.2% / d) than non-transgenic ones under normal salinity (1.4% / d). The effect of hyposaline stress on the growth of transgenic plantlets began to be seen in the second week of the experiment. The DGRs of transgenic plantlets was still high in the first week of the experiment (1 - 3.5% / d) (Fig. 7). Based on these results it is expected that, if high rainfall were to occur and seawater salinity were to drop to 15 ppt in coastal waters for 1 week, K. alvare~z' transgenic seaweed would grow normally. Moreover, under conditions of heavy rainfall for 2 - 5 weeks, transgenic seaweed would survive even with lower DGR than under normal rainfall conditions. This study showed that overexpression of the Ga gene could increase tolerance to hyposahne stress of up to 15 ppt. The mechanism of osmotic adjustment still occurs within the transgenic plantlet cells at 15 ppt salinity. This is because the Ga protein plays a role in regulating Ca2+ influx to the cytoplasm through ion channels in the plasma membrane (Gelli et al. 1997; Aharon et aL 1998; Thuleau et aL 1998; Zhang et al. 2011). In tomato plants, a constitutively active form of TGal has been shown to increase the probability of Ca2+ channels being open (Aharon et aL 1998). Ca2+ is a central regulator in plant cell physiology and plays an important role in the response to abiotic stresses including hyposaline stress (Pei & Gilroy 2008). Figure -1.0 I 1 week 0 2 weeks a3 weeks a4 weeks a5 weeks 7 Weekly DGRs of K alvareei non-transgenic and transgenic plantlets in PES medium with 15 ppt salinity Ca2+ plays a role in activating the SOS1 antiporter and the NHX antiporter. The SOSl antiporter mediates the movement of Na+ in both directions from the apoplast to the cytoplasm through secondary active transport, driven by the H+ gradient across the plasma membrane. Meanwhile, the NHX antiporter mediates the movement of Naf in both directions from the vacuole to the cytoplasm (Rodriguez-Rosales e t aL 2009). The overexpression of the Ga gene increases the Ca2+ influx to the cytoplasm in hyposaline condition, so that activation of SOSl and NHX antiporter also helps to maintain a balance of Na+ concentration between that inside and outside the cells under extreme hyposalinity (1 5 PP~). The results of calcium content analysis showed that transgenic plantlets under normal salinity conditions had higher calcium content (2.49%) than that in non-transgenic plantlets (2.06Yo) (Fig. 9). This indicated that overexpression of the Ga gene increased the Ca2+ concentration in the cytoplasrna. The transgenic plantlets in hyposaline conditions had higher calcium content (5.97% and 9.27%) than that under normal salinity (2.49%). Moreover, the calcium content of non-transgenic thalli in hyposaline conditions was lower than that under normal salinity, which was due to most of the non-transgenic thalli experiencing bleaching and being brittle in hyposaline conditions, especially at 15 ppt. Ca2+ is a structural component of cell walls and membranes in seaweed (Hurd e t a/ 2014). Increased Ca2+ concentrations in the cytoplasm caused the thalli of transgenic plantlets to thicken and become more rigid at salinity level of 15 ppt (Fig. 10B) than at 30 ppt (Fig. 10A). According to Hepler (2005), Ca2+ plays a crucial role in determining the structural rigidity of the cell wall, high content of Ca2+ should increase the rigidity and decrease the plasticity of cell walls. Ca2+ ions also play a role in regulating the flow of water into plant cells, both through apoplast and symplast (Gilliham e t al. 2011). Increased rigidity of the K al'vare@i transgenic thalli at 15 ppt salinity is an attempt to inhibit excessive influx of water into the cytoplasm under hyposalin conditions. Ion Ca2+ regulates the flow of water into the apolast by affecting the structure of the cell wall. Increased concentration of Ca2+ ions in the cytoplasm stimulates the synthesis of cell wall precursors (cellulose and nonselulose) in the cytoplasm, then these precursors are released into the apolast to form thicker or rigid cell walls (Hawkesford e t al. 2012). In the flow of water through the symplast, Ca2+ ions affect the opening of aquaporin which regulates the flow of water entering through the plasma membrane into the cytoplasm. A low concentration of Ca2+ ions in the cytoplasm will open aquaporin, while a high concentration of Ca2+ ions in the cytoplasm will close the aquaporin (Gilliham e t al. 2011). Therefore, an increase in tolerance of the transgenic plantlet to hyposalin may be due to an increase in Ca2+ ions in the cytoplasm which has closed aquaporin in the plasma membrane so as to prevent excessive inflow of water into the cytoplasm during hyposalin stress. All of the transgenic plantlet in 15 ppt were subcultured in PES medium with normal salinity. After 2 months, the entirety of transgenic plantlets thallus grew numerous thallus branches and regenerated normally (Fig. 10C). In contrast, in non-transgenic plantlets, only small part of each thallus regenerated normally, i.e., at its base and tip (Fig. 3C). Overexpression of Ga gene in k;tppqbh_Yms alvare@ transgenic plantlets - Sulistiani et al. Q Non-transgenic Transgenic 30 P P ~ 20 P P ~ 15 P P ~ Figure 9 Calcium content of K. adu~re@i non-transgenic and transgenic plantlets after 5 weeks of culture in PES medium with normal salinity (30 ppt) and hyposalinity (15-20 ppt) Figure 10 Thallus morphology of K alvare@i transgenic plantlets after 5 weeks of culture at : (A) 30 ppt, (B) 15 ppt and (C) 15 ppt + 8 weeks at 30 ppt Note: Scale bars = 7 mm. Quantification of Goc Gene Expression in Transgenic Plantlets using qPCR. Two clones of transgenic plantlets (35 and Eb), for which tolerance to hyposahity had already been proved, and one clone of non- transgenic plantlet (N) were used for expression analysis of the Ga gene using qPCR. The expression analysis was performed to determine the change in Ga gene expression in transgenic of K. aluarev? into which the Ga gene with a constitutive CaMV 35s promoter had been introduced, compared with that in non- transgenic plantlets. The changes in gene expression were analyzed by 2-MCt method. The result showed that the expression of the Gagene in the E5 transgenic plantlets increased by 6.43 times compared with expression level in non- transgenic plantlets, whereas in E6 transgenic plantlets, it increased by 8.03 times (Fig. 11). The level of transgene expression in transgenic plants is influenced by many factors, especially by the location of the integration of transgene in plant genomes, gene silencing, and promoters that accompany transgene (Page & Minocha 2004). The CaMV 35s promoter has been used as a strong and constitutive promoter for expressing transgenes in many different plants, although with different levels of efficiency depending on the species. In dicot plants, the CaMV 35s promoter is a very strong constitutive promoter, causing high levels of gene expression. The results of this study showed that CaMV 35s promoters could increase expression level of the Ga gene in transgenic plantlets of K. aluare?? (6.43 - 8.03 times) to a level hgher than that in transgenic tobacco plantlets (4.96 times) (Fajri 2015). The expression of transgenes using this promoters in transgenic plantlets of K. ahare?? has also been reported for the GFP marker gene (Rajamuddin e t al. 2014) and the H-carrageenase gene (Rajamuddin 2016). The CaMV 35s promoters have also been successfully used to express transgenes in other species of red algae, such as the GUS reporter gene in Porpbyra yexoensis (Cheney e t al. 2001) and the Lac2 reporter gene in Gradarid gracilis (Huddy e t al. 20 12). In microalga Chlamydoomonas reinhardtii, the CaMV 35s promoter had also successfully expressed the GUS reporter gene (P- glucuronidase) (Pratheesh e t al. 2012), and in microalga Dzlnaliella s a h , the promoter has successfully expressed the GFP gene reporter (Srinivasan & Gothandam 201 6). BIOTROPIA Vol. 28 No. 3,2021 1 0 N E5 E6 Figure 11 Relative expression of (2-MCt) Gcc gene in Kappaph_Ycz/s ahare@ transgenic plantlets (E5 and E6) that were tolerant of hyposalin stress, to non-transgenic (N) plantlets that were intolerant of hyposalin stress Pearson correlation analysis (Rumsey 2010) was performed on the data of relative Ga gene expression (RE) (Fig. 11) and DGRs of nontransgenic and transgenic plantlets in normal salinity (30 ppt) and hyposalinity (20 ppt and 15 ppt) (Fig. 5). The analysis showed that RE of Ga gene had a strong correlation, both with DGRs in normal salinity and hyposalinity because the correlation coefficient was more than 0.7 (Rumsey 201 0). The correlation coefficient between RE of Ga gene and DGRs at 30 ppt salinity was significant at 1% test level, whereas with DGRs at 20 and 15 ppt salinity was significant at 5% test level (Table 1). The correlation between RE of Ga gene and DGRs was linearly positive, where increased expression of the Ga gene was strongly associated with an increase in DGRs (Fig. 12). Results of correlation analysis showed that the increase in daily growth rate of transgenic plantlets, both in normal salinity and hyposalinity (Fig. 5) was strongly associated with an increased Ga gene expression in transgenic plantlets (Fig. 11). This proved that over-expression of the Ga gene had increased the growth of K. ahare?? transgenic. As reported in Arabidopsis and rice plants, Goc protein was a positive modulator of cell division processes (Ullah e t al. 2001; Chen et al 2006; Colaneri e t al 201 4; Izawa e t aL. 201 0). Table 1 Results of Pearson correlation analysis between the relative expression of the Ga gene and the daily growth rate (DGRs) in normal salinity (30 ppt) and hyposalinity (20 and 15 ppt) DGR Significance value Correlation coefficient 30 ppt salinity 0.009** 0.806 20 ppt salinity 0.020* 0.749 15 ppt salinlty 0.028* 0.724 Notes: ** = Correlation coefficient is significant at 1% test level. * = Correlation coefficient is significant at 5% test level. -1 ' Relative expression Figure 12 Correlation between the relative expression (RE) of the Ga gene with the daily growth rate of non-transgenic and transgenic plantlets on culture media with normal salinity (30 ppt) and hyposalinity (20 ppt and 15 ppt) Overexpression of Gcc gene in Kappapbycm alvareei transgenic plantlets - Sulistiani et al CONCLUSION Transgenic plantlets of K. alvare~z' had a significantly hgher daily growth rate than that of non-transgenic plantlets in PES medium with normal salinity (30 ppt) and hyposalinity (15 and 20 ppt). The transgenic plantlets showed tolerance to hyposalinity at 15 ppt. The transgenic plantlets which a high daily growth rate and tolerance to hyposalinity had a higher expression level of the Ga gene than that in non- transgenic plantlets. ACKNOWLEDGEMENTS We thank SEAMEO BIOTROP Southeast Asian Regional Center for Tropical Biology for the support of materials and facihties for mediated by the heterotrimeric G protein complex. BMC Plant Biol 14:129. Dawes CJ, Lluisma AO, Trono GC. 1994. Laboratory and field growth studies of commercial strains of Eucheuma denticulatum and Kappaph_ycus alvareei in the Philippines. J Appl Phycol6:21-4. Fajri H. 201 5. Genetic engineering of Nicotiana tabacm SR1 with Ga Gene [thesis]. Bogor OD): Institut Pertanian Bogor. Gelli A, Higgins VJ, Blumwald E. 1997. Activation of plant plasma membrane Ca2+-permeable channels by race-specific fungal elicitors. Plant Physiol 113:269-79. Gilliham M, Dayod M, Hocking BJ, Xu B, Conn SJ, Kaiser BN, ... 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