Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth

Abstract

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Figure 1

The bifunctional TPSP fusion, a transformation vector, and genomic Southern-blot hybridization of transgenic rice plants. A, The predicted amino acid sequence of the fusion boundary of TPSP is shown. The TPSP construct was made by in-frame fusion of the E. coli otsA and otsB genes, which encode TPS and TPP, respectively. B, pSB-UTPSP (Ubi1::TPSP) consists of the maize (Zea mays) ubiquitin promoter (Ubi1) linked to the TPSP coding region, the 3′ region of the potato proteinase inhibitor II gene (3′pinII), and a gene expression cassette that contains the 35S promoter, the bar-coding region, and the 3′ region of the nopaline synthase gene (nos). The restriction enzymes, the expected fragment sizes, and the hybridization probe (probe) used for genomic DNA-blot analyses are shown below the map. C, Genomic Southern-blot analysis of Ubi1::TPSP transgenic rice plants. Genomic DNAs from the leaves of five Ubi1::TPSP plant lines and from untransformed control plants (NT) were digested with EcoRI (RI) or SacI (Sc), fractionated on an agarose gel, blotted onto a nylon membrane, and hybridized with the probe for TPSP coding region (described in B).

Figure 2

Transcript levels of TPSP and stress-inducible rice genes in the leaves of Ubi1::TPSP and untransformed plants. A, Northern-blot analysis was performed using total RNA from young leaves of five Ubi1::TPSP plant lines (shown in Fig. 1C) and from untransformed control plants (NT). The blots were hybridized with probes for TPSP (as described in Fig. 1B), Lip5 (Aguan et al., 1991), and Dip1 (GenBank accession no. AU095986). Equal loading of total RNA samples was verified by reprobing the membrane with the rice rbcS gene for Rubisco (Kyozuka et al., 1993). Transcript levels of TPSP and Lip5 in the Ubi1::TPSP lines were calculated using those of corresponding rbcS as a reference and the resultant values were then normalized to 1 for that from NT. B, Northern blots of total RNA from untransformed plants immediately before and after stress treatments. The blots were hybridized with probes for Lip5, Dip1, and rbcS. Transcript levels of rbcS were previously reported to be decreased upon exposure to drought and salt stresses (Weatherwax et al., 1996). For drought stress, 14-d-old seedlings were air dried for 2 h at 28°C; for salt stress, 14-d-old seedlings were exposed to 400 mm NaCl for 2 h at 28°C. All of the experiments were carried out under continuous 150 μmol m² s⁻¹ light conditions. Ethidium bromide (EtBr) staining of total RNA was used to ensure equal RNA loading.

Figure 3

HPIC analysis of trehalose accumulation in Ubi1::TPSP plants. A, The chromatograms show carbohydrate profiles from a standard containing 1 μg of trehalose (T), leaf and seed extracts that were prepared from untransformed controls (NT), and two transgenic lines (Ubi1::TPSP-1 and -2). B, Carbohydrate profiles from a standard containing 1 μg each of trehalose (T), Glc (G), Suc (S), maltose (M), T-6-P, and Glc-6-phosphate (G-6-P), leaf extracts that were prepared from untransformed controls (NT), and three transgenic lines (Ubi1::TPSP-3, -4, and -5).

Figure 4

Growth phenotypes of T₂ plants of Ubi1::TPSP-1 and untransformed control plants (NT), 3 d after germination (3 DAG), 7 d after germination (7 DAG), 14 d after germination (14 DAG), and in mature plants setting seeds (mature).

Figure 5

Stress tolerance of T₂ plants of Ubi1::TPSP-1 and untransformed control plants (NT). A, Six-day-old seedlings were grown in the greenhouse for 10 d (10 D) and 12 d (12 D) after watering stopped. Photos of the upper leaves of corresponding plants are shown at either side of the figures. B, For drought stress, 14-d-old seedlings were air dried for 1 h at 28°C; for salt stress, 14-d-old seedlings were exposed to 150 mm NaCl for 2 h at 28°C; and for cold stress, 14-d-old seedlings were exposed to 4°C for 6 h. All of the experiments were carried out under continuous 150 μmol m² s⁻¹ light conditions. Chlorophyll fluorescence (variable fluorescence [F_v] and maximal fluorescence [F_m]) was measured using a pulse modulation fluorometer. Six seedlings were measured and averaged for each treatment protocol.

Figure 6

Salt tolerance of non-transgenic and Ubi1::TPSP seedlings grown in the presence of 100 mm NaCl. Ten T₂ seeds from each of the five (1–5) Ubi1::TPSP lines and the non-transgenic (NT) plants were germinated and grown in hydroponic solutions that contained 100 mm NaCl under continuous 150 μmol m² s⁻¹ light conditions. A, The shoot length was scored at various intervals. Each data point represents the mean ± se of triplicate experiments (n = 10). B, Representative seedlings at 10 d after germination are shown.