Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity

Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity 1 3 first-author 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xueqingzhong@hotmail.comzstressc@online.sh.cn †E-mail:xueqingzhong@hotmail.comzstressc@online.sh.cn xueqingzhong@hotmail.com 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xueqingzhong@hotmail.comzstressc@online.sh.cn †E-mail:xueqingzhong@hotmail.comzstressc@online.sh.cn xueqingzhong@hotmail.com Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Science, Shanghai 200032, China Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Science, Shanghai 200032, China School of Resource and Environment Management, Guizhou College of Finance and Economics, Guiyang 550004, China School of Resource and Environment Management, Guizhou College of Finance and Economics, Guiyang 550004, China Transgenic Brassica compestris L. spp. chinensis plants expressing a choline oxidase ( codA ) gene from Arthrobacter globiformis were obtained through Agrobacterium tumefaciens -mediated transformation. In the transgenic plants, codA gene expression and its product transportation to chloroplasts were detected by the enzyme-linked immunosorbent assay (ELISA) examination, immunogold localization, and 1 H-nuclear magnetic resonance ( 1 H-NMR). Stress tolerance was evaluated in the T 3 plants under extreme temperature and salinity conditions. The plants of transgenic line 1 (L1) showed significantly higher net photosynthetic rate ( P n ) and P n recovery rate under high (45 °C, 4 h) and low temperature (1 °C, 48 h) treatments, and higher photosynthetic rate under high salinity conditions (100, 200, and 300 mmol/L NaCl, respectively) than the wild-type plants. The enhanced tolerance to high temperature and high salinity stresses in transgenic plants is associated with the accumulation of betaine, which is not found in the wild-type plants. Our results indicate that the introduction of codA gene from Arthrobacter globiformis into Brassica compestris L. spp. chinensis could be a potential strategy for improving the plant tolerance to multiple stresses. Brassica compestris L. spp. chinensis codA Stress Glycine betaine Net photosynthetic rate ( P n ) Introduction 1 Glycine betaine (betaine) is a quaternary ammonium compound that occurs naturally in a wide variety of plants, animals, and microorganisms. It can stabilize the structure of proteins, and maintain the integrity of cell membrane, thus enhancing the tolerance of organisms to high salt, high temperature, and cold injuries (Gorham, 1995 1995 ). Many plant species, including rice, tomato, tobacco, and Arabidopsis, are not able to synthesize betaine, but may be engineered to accumulate betaine through a transgenic approach to improve their tolerance to various extreme conditions (Sakamoto and Murata, 2001 2001 ; Prasad and Saradhi, 2004 2004 ). The first successful transformation of a plant for the synthesis of betaine in Arabidopsis thaliana was reported by Hayashi et al. ( 1997 1997 ). Within chloroplasts, choline oxidase encoded by the codA gene produces betaine, which provides leaf protection from high temperature and other stresses in Arabidopsis thaliana (Alia et al., 1998a 1998a ; Sakamoto and Murata, 2000 2000 ). Higher betaine accumulation has been reported to endow higher salt tolerance in plants (Saneoka et al., 1995 1995 ). For example, transgenic Arabidopsis expressing codA exhibits enhanced germination under high salinity conditions (Hayashi et al., 1998 1998 ). Transgenic tobacco ( Nicotiana tabacum ) plants show improved tolerance to salt and drought stresses (Holmström et al., 2000 2000 ; Huang et al., 2000 2000 ; He et al., 2001 2001 ). Transgenic rice is advantageous in maintaining stable osmotic pressure and promoting roots growth, thus enhancing the plant tolerance to water deficit (Sakamoto et al., 1998 1998 ; Sawahel, 2003 2003 ). In the transgenic codA plants, the enhanced performance in germination and tolerance to various stress conditions has been associated with the accumulation of betaine (Alia et al., 1998a 1998a ; 1998b 1998b ; 1999 1999 ; Prasad and Saradhi, 2004 2004 ). Moreover, it has been found that the transgenic codA plants show a significantly lower trend of electrolyte leakage, as well as hydrogen peroxide and malondialdehyde contents than wild-type plants (Parvanova et al., 2004 2004 ). Brassica compestris L. spp. chinensis is a vegetable crop widely cultivated in South China. It does not synthesize betaine in vivo, and is sensitive to salt, drought, and high temperature stresses. Via the Agrobacterium tumefaciens -mediated transformation procedure, we have successfully transferred the codA gene into the genome of Brassica compestris L. spp. chinensis var. Aikangqing. In this study, we evaluated the transgenic plants for their tolerance to high and low temperatures and high salinity stresses by examining their photosynthetic performance at the growth stage. We report here the enhanced tolerance observed in these transgenic plants. Materials and methods 1 Construction of vector plasmids 2 The binary vector pGAH/codA (a kind gift from Dr. Norio MURATA), harboring an expression cassette of the codA gene from Arthrobacter globiformis , was used for transformation. The construct contains the 35S promoter of cauliflower mosaic virus, the codA gene, and the terminator for nopaline synthase gene (Fig. 1 1 ). Its T-DNA region also contains genes encoding a neomycin phosphotransferase (NPTII), which confers kanamycin resistance, and a hygromycin phosphotransferase (HPT), which confers hygromycin resistance. In order to direct the expressed choline oxidase to chloroplasts, where betaine can be accumulated to a greater degree, the construct was modified by adding a complementary DNA (cDNA) sequence encoding the transit peptide of a tobacco Rubisco subunit to the codA coding region. The resultant binary vector plasmid was introduced into A. tumefaciens strain EHA101 containing the Ti plasmid, which was then used for plant transformation. RB: right border; Km r : kanamycin resistance gene; 35S pro: the 35S promoter of cauliflower mosaic virus; rbcS tr: the sequence that encodes transit peptide of subunit of Rubisco from tobacco; codA : the gene encoding choline oxidase (COD); NOS ter: NOS terminator; P1 and P2 are upstream and downstream primers, respectively; Hyg r : hygromycin resistance gene; LB: left border Transformation of Brassica compestris L. spp. chinensis 2 After germinating for 5–7 d, the hypocotyls or cotyledons of Brassica compestris L. spp. chinensis were pre-cultured for 1 d on the differentiation medium. A. tumefaciens strain EHA101 harboring the pGAH/codA plasmid was grown at 28 °C for 36 h in a 250-ml flask containing 50 ml of yeast extract broth (YEB) liquid medium supplemented with kanamycin 50 mg/L and hygromycin 50 mg/L until the late exponential phase. The explants pre-cultured for 1 d were co-cultured with the A. tumefaciens for 5 min, and transferred onto a differentiation Murashige and Skoog (MS) medium supplemented with 0.5 mg/L indole-3-acetic acid (IAA), 0.025 mg/L naphthaleneacetic acid (NAA), 5 mg/L 6-benzyladenine (6-BA), 300 mg/L carbenicillin, and 15 mg/L kanamycin. After co-incubation with A. tumefaciens for 2 d at 25 °C (14-h light period) and incubation without A. tumefaciens for 4–5 weeks, the shoots of the explants were excised and transplanted to the root induction medium containing kanamycin. The surviving explants were carefully transferred into plastic pots (10 cm×10 cm×10 cm) loaded with sterilized vermiculite, and grown in a phytotron with fixed temperature (25 °C), 16-h light period (350–400 μmol/(m 2 ∙s)), and a relative humidity of 70%–80%. Transformants were obtained from the selection medium with 60 mg/L kanamycin. Development of subsequent T 1 , T 2 , and T 3 plants 2 T 1 seeds harvested from the T 0 transgenic plants were first washed in tap water for 3 h, soaked in 1.5% (w/v) Xiaojieling solution for 30 min and in 0.1% (w/v) HgCl 2 solution for 2–3 min, and washed repeatedly in sterile water. The treated seeds were sowed in the MS basal (MS 0 ) medium containing 60 mg/L kanamycin for testing and selecting T 1 plants for antibiotic resistance. The same treatment described above was also applied to T 2 seeds. Those plants without segregation for antibiotic resistance were considered as homozygous. The T 3 seeds harvested from self-crossed T 2 plants were used for subsequent stress experiments. Polymerase chain reaction (PCR) examination of the transgenic plants 2 To detect the presence of the codA gene in the transgenic plants, a pair of primers was designed (P1: 5′-AACATCGAGAACCTGAGCGACAGG-3′; P2: 5′-AGCATCAACAGCTTCGGCGTATC-3′) using codA as template and used in the PCR. Each 25 μl PCR solution consisted of 10 pmol/L of each primer, 50 μmol/L of deoxyribonucleoside triphosphate, 2.5 μl 10× Taq buffer, 1 μl extracted DNA solution, 1 U Taq enzyme, 1% (v/v) dimethyl sulphoxide (DMSO), and 5% (v/v) glycerine. Assays were performed on the MJ Research Minicycler (Watertown, MA) with the following thermocycle profile: 94 °C for 5 min, 40 amplification cycles of 94 °C for 1 min, 59 °C for 1 min, 72 °C for 1 min, and finally 72 °C for 10 min. Enzyme-linked immunosorbent assay (ELISA) examination of the transgenic plants 2 Fresh leaves of Brassica compestris L. spp. chinensis were ground in liquid nitrogen. The powder was suspended with a ratio of 3 ml/g fresh weight (FW) in a protein extraction buffer containing 0.1 mol/L Tris-HCl (pH 8.0), 0.01 mol/L MgCl 2 , 18% (w/v) sucrose, and 40 mmol/L 2-mercaptoethanol, and cell debris were removed by centrifugation at 10 000× g for 15 min at 4 °C. The antigen supernatant containing the crude protein extraction of choline oxidase was subjected to ELISA examination, as previously described by Kramer et al. ( 1995 1995 ) with some modification. After 50 μl of antigen solution was moved from the incubated wells and washed three times, nonspecific protein used in blocking buffer was incubated for 2 h at room temperature. After discarding the blocking buffer and triple washing, the 50 μl primary antibody (rabbit immune body of choline oxidase) with a 1/200 (v/v) dilution in blocking buffer was added into each well and incubated for 2 h at room temperature. Following triple washing with sterilized water, 50 μl of goat anti-rabbit immunoglobulin G (IgG)-alkaline phosphatase (AP) conjugate (Sino-American Biotechnology Co., China) with a 1/1000 (v/v) dilution in blocking buffer was added into each well and incubated for 2 h at room temperature. After triple washing, dye conversion was initiated by charging the wells with 50 μl of the para -nitrophenylphosphate (pNPP) at 37 °C for 1 h, to determine if choline oxidase existed in the transgenic plants. The reaction was terminated by adding 25 μl 0.5 mol/L NaOH. Immunological examination of the expressed choline oxidase 2 The localization of the expressed choline oxidase was examined immunocytochemically as previously described by Mustardy et al. ( 1990 1990 ) with some modifications. For preparation of a soluble fraction, small pieces (2 mm×2 mm) of five-leaf-stage leaves of the transgenic and non-transgenic plants were cut and fixed in 2% (v/v) glutaraldehyde overnight at 4 °C and in 1% (w/v) osmium tetroxide for 2 h at 4 °C. Following triple washing with 5% (w/v) phosphate buffer solution (PBS), 20 min each time, samples were dehydrated in serial concentrations of ethanol (50%→70%→80%→90%→95%→100%) and 100% acetone, infiltrated overnight at room temperature in an acetone-Epon 812 mixture (1:1, v/v), embedded in fresh Epon 812, and subjected to heat-polymerization for 12 h at 37 °C, 12 h at 45 °C, and 24 h at 60 °C. Ultrathin sections (~60 nm) were cut with ultramicrotomy (speed 2–3 mm/s). Antibodies against choline oxidase raised in a rabbit were purchased commercially from Sigma (St. Louis, MO, USA). Initially, the ultrathin sections were mounted on uncoated nickel grids, rinsed with 5% H 2 O 2 and PBS buffer for three times (5 min per time), subsequently treated for 1 h at 25 °C with a blocking solution consisting of 0.25% (w/v) bovine serum albumin (BSA) (fraction V, Sigma), 0.05% (v/v) Tween 20 (Bio-Rad), 0.05% (w/v) NaN 3 , 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.17 mol/L H 3 BO 4 , and 0.12 mol/L NaCl (pH 8.5), and then exposed to the primary antiserum diluted in 1:200 (v/v) in PBS buffer and incubated for 30 min. This was followed by 30 min incubation with the 10 nm-gold-conjugated secondary antiserum (goat anti-rabbit) in a 1:20 (v/v) dilution in PBS containing 1% (w/v) BSA. Samples were required to be vigorously rinsed six times with 1% PBS between the incubations, 5 min per time for optimum labeling, and finally were rinsed in sterilization ddH 2 O. After dried in the air, ultrathin sections were stained with 4% (v/v) uranyl acetate for 15 min and 2% (w/v) lead citrate for 5 min, and then examined in a transmission electron microscope (H-600-4, Hitachi Ltd., Japan). Betaine measurement 2 Approximately 5 g fresh leaf materials were ground to a fine powder in liquid nitrogen. The powder was suspended in 25 ml of 1.0 mol/L H 2 SO 4 and incubated at 25 °C for 2 h. Cell debris were removed by centrifugation at 1000× g for 10 min. The supernatant was incubated in 10 ml of KI-I 2 solution (15.7 g I 2 and 20 g KI dissolved in 100 ml of 1 mol/L HCl) at 0 °C for 2 h, and centrifuged at 1000× g for 30 min and the periodide adducts of betaine were collected. The resulting periodide adducts of betaine were then dissolved in 0.5 ml of D 2 O, which contained 0.5 mmol 2-methyl-2-propanol (tBA) as an internal standard, and were used for the determination of 1 H-nuclear magnetic resonance ( 1 H-NMR). Detailed procedure can be found in Hayashi et al. ( 1997 1997 ). Net photosynthetic rate ( P n ) measurement 2 The plants of 6–8-leaf stage were treated with low temperature (1 °C for 48 h) or high temperature (45 °C for 4 h), then moved into the phytotron to recover for 1 h, and measured for P n with a portable CO 2 gas analyzer (Model CI-301, CID Inc., Vancouver, WA, USA). The antepenultimate leaves were selected for the P n measurement. According to the method of Yang ( 1999 1999 ), 10 leaf disks from the leaves, which were measured for photosynthesis, were used to analyze the contents of chlorophyll a and b (mg/g FW). All measurements were done with three replicates. Salt tolerance 2 At 8–9-leaf stage, 20 plants of T 3 generation from the transgenic lines 1 and 2 (L1 and L2) and wild-type were subjected to salt treatments by adding NaCl to the growth medium with 50 mmol/L increments every 24 h until a final concentration of 0, 100, 200, and 300 mmol/L, respectively, and maintained for 7 d (Xu et al., 2001 2001 ). The survival rate in each treatment was analyzed to compare the salt tolerance between transgenic and wild-type plants, and the antepenultimate leaves were measured for P n . The survival rate ( Su ) was evaluated by Su (%)= N s / N a ×100 , where N s is the number of survival plants per treatment, and N a is the number of all plants per treatment. Results 1 Transformation with the codA gene 2 We used 769 Brassica compestris L. spp. chinensis explants (339 cotyledons and 430 hypocotyls) to conduct the transformation experiments with the codA gene carried by the vector plasmid shown in Fig. 1 1 , and eventually obtained 129 transgenic plants (T 0 ), which were derived from 45 cotyledons and 84 hypocotyls. The differentiation rates for the cotyledon and hypocotyl in our experiments were extremely high (13.27% and 19.53%, respectively). We have developed an efficient codA transformation system for Brassica compestris L. spp. chinensis through screening of the compositions of differentiation and selection media (data not shown). From the 129 transgenic plants (T 0 ), we obtained 10 transgenic plants (T 1 plants) showing resistance to both kanamycin and hygromycin. Subsequent T 2 and T 3 plants were obtained by the procedure as described in Section 2.3. In each generation from T 0 to T 3 , the presence of the transgene ( codA ) was confirmed by PCR (Fig. 2 2 ). The band with same molecular weight ( M W ) size amplified in L1 and L2 plants was also amplified from the plasmid pBinMoBc, which was used as the positive control, but no bands appeared in the wild-type plants, suggesting that the codA has been successfully inserted into the Brassica compestris L. spp. chinensis genome by A. tumefaciens -mediated transformation. Lane 1: DNA marker which has been purchased commercially (TaKaRa); Lane 2: transgenic line 1 (L1); Lane 3: transgenic line 2 (L2); Lane 4: PCR fragment of plasmid pGAH/codA; Lane 5: wild-type plant We further evaluated the existence of the choline oxidase, the product of the codA gene through ELISA examination of the leaves of L1 and L2 plants. The results showed that only L1 and L2 plants had the positive reaction (Fig. 3 3 ), indicating that the coda gene had been efficiently expressed in each line of the transgenic plants, and that the expressed precursor had been effectively processed to the mature protein. Lane 1: negative control (substrate+buffer+water); Lane 2: non-transgenic wild-type plant; Lane 3: transgenic line 1 (L1); Lane 4: transgenic line 2 (L2) To determine the localization of the expressed choline oxidase, the immunogold labeling technique was applied in the transgenic and wild-type plants. Many colloid gold particles were observed in the chloroplasts of the L1 and L2 plants with the immunoelectron microscope (Figs. 4b and 4c 4b and 4c ). In contrast, the density of gold particles in the chloroplasts of the wild-type plants was at the background level (Fig. 4a 4a ). The results confirm that choline oxidase was located truly in the chloroplasts of the transgenic plants. (a) Wild-type plant; (b) Transgenic line 1 (L1) plant; (c) Transgenic line 2 (L2) plant Betaine accumulation 2 The results from the 1 H-NMR spectrometry experiment showed that there were characteristic peaks of betaine with accumulation levels of 0.224 μmol/g FW and 0.221 μmol/g FW in the transgenic L1 and L2 plants, respectively, but no betaine accumulation in the wild-type plants (Fig. 5 5 ). Therefore, the results suggest that the transgene, codA , expressed well and the gene product, choline oxidase, functioned properly in the transgenic plants. (a) Wild-type plant; (b) Transgenic line 1 (L1) plant; (c) Transgenic line 2 (L2) plant. GB: glycine betaine; tBA: 2-methyl-2-propanol or tert-butyl alcohol, which is similar structurally to betaine and used as an internal standard for the estimation of the content of betaine. The distance between the small peaks was indicated by coupling constants. δ : chemical shift Comparison of photosynthesis under low temperature stress 2 We measured the net photosynthetic rate ( P n ) at the time point of 1 h after the 6–8-leaf stage plants were exposed to low-temperature (1 °C) for 48 h. The transgenic L1 and L2 plants showed a significantly higher P n (9.61 μmol/(m 2 ∙s) and 9.22 μmol/(m 2 ∙s), respectively) than the wild-type control plants (5.19 μmol/(m 2 ∙s)) ( P < 0.01), as shown in Table 1 1 . Values were expressed as mean±SD ( n =3) Significant differences at P < 0.01 Significant differences at P < 0.001 L1, L2: transgenic lines 1 and 2, respectively Type P n (μmol/(m 2 ∙s)) E (mmol/(m 2 ∙s)) Sr ((m 2 ∙s)/mol) Wild 5.19±1.07 2.45±0.27 28.18±1.41 L1 9.61±0.83 ** ** 2.52±0.26 13.00±1.61 *** *** L2 9.22±0.51 ** ** 2.49±0.21 13.08±0.16 *** *** Under the same stress condition, stoma resistance ( Sr ) of the transgenic plants was lower than that of wild-type plants, that is, CO 2 entering into a mesophyll cell through the stomata is reinforced by increased gradient of CO 2 between the external atmosphere and the intercellular space inside the transgenic plant leaves because of the enhanced photosynthesis in the transgenic plants (Table 1 1 ). We also measured the chlorophyll contents in the different plants and the results showed that transgenic L2 plants had higher chlorophyll a/b ratio than the wild-type plants ( P < 0.05) (Table 2 2 ). Values were expressed as mean±SD ( n =3) Significant difference at P < 0.05 L1 and L2 are the transgenic lines 1 and 2, respectively 2 Type 4 Chlorophyll value (mg/g FW) a b ab a/b Wild 0.4251±0.0245 0.1765±0.0113 0.6016±0.0351 2.408±0.1134 L1 0.4154±0.0211 0.1677±0.0089 0.5831±0.0255 2.477±0.1225 L2 0.4469±0.0231 0.1487±0.0067 0.5956±0.0348 3.005±0.1317 * * Comparison of recovery of net photosynthetic rate ( P n ) under high temperature stress 2 Under non-stressed condition, the P n in the transgenic line L1 (5.36 μmol/(m 2 ·s)) was almost the same as the wild-type control plants (5.34 μmol/(m 2 ·s)). However, after 4 h exposure to high temperature (45 °C) and 1-h restoration in the phytotron, the P n of the transgenic line L1 dropped to 4.01 μmol/(m 2 ·s), which is significantly higher than that of the wild-type plants (1.87 μmol/(m 2 ·s)) (Table 3 3 ). Nevertheless, after 25-h restoration, there is no significant difference for the P n between them (Table 3 3 ). The results suggest that the transgenic plants can recover much faster from the stress than the wild-type plants (74.9% vs. 35.0% with 1-h restoration). Values were expressed as mean±SD ( n =3); A total of 15 plants per treatment were tested Significant difference at P < 0.05 Significant difference at P < 0.01 L1: transgenic line 1 2 Type 3 P n (μmol/(m 2 ∙s)) 2 Recovery of P n (%) Pre-exposure 1 h after exposure 25 h after exposure 1 h after exposure 25 h after exposure Wild 5.34±0.37 1.87±0.1 4.02±0.38 35.0±1.0 75.2±12.8 L1 5.36±0.42 4.01±0.30 ** ** 4.85±0.55 74.9±11.0 ** ** 90.5±3.7 * * Comparison of survival rate and photosynthesis under salt stress 2 The survival rate for the transgenic L1 plants was higher than that of wild-type plants under all the three levels of NaCl concentration (Table 4 4 ). For example, under 100 mmol/L NaCl, 94.4% of L1 plants vs. 65.4% of wild-type plants survived (Fig. 6 6 ). While most of the wild-type plants (16.7% survivals) died under 200 mmol/L NaCl, more than half (53.3%) of the transgenic L1 plants were still alive. Though most died among both types of plants under 300 mmol/L NaCl, there were still 27.8% transgenic L1 plants that survived. (a) Transgenic L1 plants; (b) Wild-type plants; (c) Transgenic plants with flowering Values were expressed as mean±SD ( n =3) Significant difference at P < 0.05 Significant difference at P < 0.01 Significant difference at P < 0.001 P n : net photosynthetic rate; E : evaporation rate; Sr : stoma resistance; Su : survival rate NaCl (mmol/L) Types P n (μmol/(m 2 ∙s)) a E (mmol/(m 2 ∙s)) Sr ((m 2 ∙s)/mol) Su (%) 0 Wild 5.71±0.67 1.28±0.11 27.72±2.74 100±0 L1 5.75±0.68 0.95±0.14 29.71±3.01 100±0 100 Wild 1.50±0.53 0.61±0.12 61.66±3.87 65.4±8.9 L1 4.12±0.75 ** ** 0.87±0.13 42.21±3.65 * * 94.4±8.0 ** ** 200 Wild 0.27±0.41 0.40±0.09 84.11±3.23 16.7±7.8 L1 2.66±0.52 *** *** 0.74±0.13 * * 47.75±3.90 * * 53.3±9.1 *** *** 300 Wild −0.10±0.12 0.37±0.08 90.93±4.22 13.6±6.0 L1 1.27±0.21 *** *** 0.58±0.11 * * 72.45±4.43 * * 27.8±5.7 *** *** Similar P n were observed between the transgenic line L1 plants (5.75 μmol/(m 2 ∙s)) and wild-type plants (5.71 μmol/(m 2 ∙s)) under non-stress condition. However, like the situation discussed above in high temperature stress, less reduction of P n was also observed in the transgenic L1 plants than in the wild-type plants under all the three NaCl concentrations (Table 4 4 ). For example, under 100 mmol/L NaCl, the P n of the transgenic L1 plants was close to three-fourths of that observed under non-stress condition, but that was only one-fourth for the wild-type control plants (1.50 μmol/(m 2 ∙s)). As for the evaporation rate ( E ), no differences were observed between the transgenic and wild-type plants under non-stress condition. However, the evaporation rate was higher in the transgenic plants than in the wild-type plants under both 200 and 300 mmol/L NaCl ( P < 0.05) (Table 4 4 ). Under high salinity stress, the transgenic plants showed a lower stoma resistance ( Sr ) than the wild-type plants (Table 4 4 ), indicating that the high photosynthesis for transgenic plants is due to less reduced stomata opening from stress, i.e., the stomata opening of transgenic plants is less affected by salt stress than that of wild-type plants, which facilitates CO 2 entering the leaf and increases photosynthesis. Therefore increased photosynthesis is the result of more CO 2 entering inside the leaf through stomata. Discussion 1 Brassica compestris L. spp. chinensis is a very important vegetable crop, commonly grown in South China. It is not able to synthesize endogenously betaine, and therefore is very sensitive to salt, drought, high temperature, and other environmental stresses. In this study, we obtained transgenic plants expressing codA gene from Arthrobacter globiformis and demonstrated enhanced tolerance to extreme temperature and high salinity in these transgenic plants. The content of betaine in the transgenic plant is influenced directly by its subcellular location. In tomato, there are reports that the transcript of codA could be targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). A comparison between these three types of transgenic plants showed that Chl-codA plants with the lowest amounts of betaine exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses, suggesting more effectiveness of chloroplastic betaine in protecting plant cells (Park et al., 2007 2007 ). Our results showed that the codA gene product in Brassica compestris L. spp. chinensis was targeted to chloroplasts, which agreed with the results from both A. thaliana (Alia et al., 1998b 1998b ) and Brassica juncea (Prasad et al., 2000 2000 ). The contents of betaine in chloroplasts of the codA transgenic plants varied generally due to the different plant species, such as 0.30 μmol/g FW in transgenic Diospyros kaki and Lycopersicon esculentum , and 1.43 μmol/g FW in transgenic Solanum tuberosum (Chen and Murata, 2008 2008 ). Moreover, the expression level of codA is affected by the cultivation conditions and the tested tissues or organs for the transgenic plants. In our experiment, all the plants were cultured in water, and thus had much higher water content (~90%) than that cultured in soil. Therefore, our transgenic plants had low measured values of betaine (~0.22 μmol/g FW). However, they still showed high tolerance to extreme temperature and high salinity. Environmental stresses such as salt and extreme temperature will eventually cause yield losses for plant production. During adaptation, plant may develop tolerance to environment stresses by accumulating some small organic solutes, including the polyhydroxylated compounds, carbohydrate, amino acid, betaine, and related compounds (Bohnert and Jensen, 1996 1996 ; Hayashi et al., 1998 1998 ) known as compatible materials (Bohnert et al., 1995 1995 ). In plants, the major compatible osmoprotectant solutes include betaine, proline, and polyols (Rontein et al., 2002 2002 ). In this study, transgenic plants expressing the codA gene were able to accumulate betaine in vivo, which may have possible protective effects on the biological macromolecules (Schobert, 1977 1977 ). In the chloroplast of spinach and sugar beet plants, betaine could be synthesized and accumulated naturally, but not in that of most other plant species. However, we could engineer theses plants to accumulate betaine through a transgenic approach (Sakamoto and Murata, 2001 2001 ; Prasad and Saradhi, 2004 2004 ). The strategy for engineering betaine synthesis was employed by transformation with the codA gene which encodes choline oxidase and offers an attractive conversion from choline to betaine under the enzymatic catalysis. Transgenic A. thaliana plants with the codA gene significantly enhanced the tolerance to low temperature and high-salt stress (Hayashi et al., 1998 1998 ). Under low temperature, the transgenic A. thaliana with codA gene had an obviously higher biological output than the wild-type control (Hayashi et al., 1997 1997 ; Alia et al., 1999 1999 ). The codA -mediated tolerance to salt stress has been reported in other field crops, including corn, (Saneoka et al., 1995 1995 ), rice (Mohanty et al., 2002 2002 ; Sawahel, 2003 2003 ), Brassica juncea (Prasad et al., 2000 2000 ), and tobacco (He et al., 2001 2001 ). Here we reported that transgenic Brassica compestris L. spp. chinensis seedlings expressing a codA gene from A. globiformis could accumulate betaine in vivo and showed significant tolerance to high and low temperatures and high salinity stresses, compared with wild-type plants. Moreover, the wild-type plants had more difficulty surviving compared to the transgenic plants under the 300 mmol/L NaCl condition, suggesting that betaine may be important for osmotic adjustment under salinity stress in the transgenic plants. It has been demonstrated that the biosynthesis of betaine is stress-inducible (Sakamoto and Murata, 2002 2002 ). The direct protective effects of betaine on macromolecules and membranes may be not only osmotic, but also via the mechanism of compatible solutes and oxygen radical scavenging (Blumwald and Grover, 2006 2006 ). This point may be supported by our results that the transgenic seedlings of Brassica compestris L. spp. chinensis showed a higher P n after the low temperature treatment and a higher recovery rate of photosynthesis after exposure to high temperature (45 °C for 4 h). The role of betaine in stress tolerance could be protecting the oxygen-evolving PSII complex, stabilizing the protein structure of PSII complex, and maintaining ATP synthesis under stress conditions (Sakamoto and Murata, 2001 2001 ; Rahman et al., 2002 2002 ). In this study, we found that the transgenic plants with codA gene showed higher P n and lower stoma resistance accompanying with higher evaporation rate than the wild-type plants under the salt stress condition. In contrast, under temperature stress, an insignificant difference of evaporation rate but higher P n was observed in the transgenic plants, compared with the wild-type plants. This may indicate that the action mechanisms for increasing P n were different in the transgenic plants under the two different stress conditions. The stomatal resistance may be involved in the salt stress, but not in the temperature stress. Our results are consistent with the experiment of Mäkelä et al. ( 1998 1998 ), who showed that application of exogenous betaine can significantly increase stomatal conductance of plants grown in saline conditions. An increase in stomatal conductance was related to the maintenance of higher turgor pressure or water potential in plant leaf cells (Cushman et al., 1989 1989 ). We found that the transgenic plants with codA did not show significantly higher transpiration rates than the wild-type plants in the temperature stress experiments, indicating a less or insignificant effect of stomatal opening on the P n . Actually, the P n can be affected by a number of factors, including a variety of antioxidant enzymes in vivo, such as superoxide dismutase, catalase, peroxidase, ascorbic acid oxidase, and glutathione reductase. The activity of those enzymes may be well conserved by the presence of betaine. Some experiments showed that the increase of the above antioxidant enzymes could efficiently eliminate active oxygen and oxygen free radicals, and thus maintain the structural stability and integrity of the cell membrane and chloroplast membrane under stress conditions (Hayashi et al., 1997 1997 ). It appears that the remaining higher stomatal conductance under salt stress and protecting integrity of photosynthetic apparatus under temperature stress might be two reasonable explanations for increased photosynthesis observed in our transgenic plants. References Alia , H Hayashi , A Sakamoto , N Murata . 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Modern Experiment Protocol in Plant Physiology , 1999 . : Beijing, China Science Press , 95 - 96 . (in Chinese) . Determination of Chlorophyll Content We thank Dr. Norio MURATA (National Institute for Basic Biology, Okazaki, Japan) for his kind gift of the binary vector pGAH/codA and are grateful to Mr. Fang-zheng WANG, Prof. Da-quan XU, Ms. Ya-fang ZHU, and Mr. Ji-hu SU (Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, China) for technical assistance. We also thank Dr. Neng-yi ZHANG (Cornell University, USA) for English language correction. 10.1631/jzus.B1000137 Project supported by the National Science Foundation of China (No. 30571146) and the National Key Basic Research Special Foundation of China (No. G1999011700) 2010-04-15 2010-09-28 2010-05-18 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 11