Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress

Abstract

The binding protein (BiP) is an important component of endoplasmic reticulum stress response of cells. Despite extensive studies in cultured cells, a protective function of BiP against stress has not yet been demonstrated in whole multicellular organisms. Here, we have obtained transgenic tobacco (Nicotiana tabacum L. cv Havana) plants constitutively expressing elevated levels of BiP or its antisense cDNA to analyze the protective role of this endoplasmic reticulum lumenal stress protein at the whole plant level. Elevated levels of BiP in transgenic sense lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination and tolerance to water deficit during plant growth. Under progressive drought, the leaf BiP levels correlated with the maintenance of the shoot turgidity and water content. The protective effect of BiP overexpression against water stress was disrupted by expression of an antisense BiP cDNA construct. Although overexpression of BiP prevented cellular dehydration, the stomatal conductance and transpiration rate in droughted sense leaves were higher than in control and antisense leaves. The rate of photosynthesis under water deficit might have caused a degree of greater osmotic adjustment in sense leaves because it remained unaffected during water deprivation, which was in marked contrast with the severe drought-induced decrease in the CO(2) assimilation in control and antisense leaves. In antisense plants, the water stress stimulation of the antioxidative defenses was higher than in control plants, whereas in droughted sense leaves an induction of superoxide dismutase activity was not observed. These results suggest that overexpression of BiP in plants may prevent endogenous oxidative stress.

Figure 1

Schematic diagram of the chimeric BiP constructs transformed into tobacco via pBI121-derived binary vector. The soy BiP gene in the sense (A) and antisense (B) orientation was placed under the control of the constitutive CaMV 35S promoter (35S-P) and the 3′ nos polyadenylation signal (3′nos). The nptII gene expression is driven by the nos promoter (nos-P). LB and RB correspond to the T-DNA left and right borders, respectively. The positions of some restriction enzyme sites are indicated.

Figure 2

Enhanced levels of BiP in transgenic tobacco plants. Equivalent amounts of total protein (30 μg per lane) extracted from the fully expanded third leaf of untransformed, wild-type plant (lane 1), pBI121-trasnformed control plant (lane 2), four independent transgenic 35S-BiPAS (antisense) tobacco plants (lanes A5, A10, A15, and A16), and four independent transgenic 35S-BiPS (sense) tobacco plants (lanes S1, S3, S6 and S7) were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with an anti-soybean BiP antibody. Different numbers after S and A symbols indicate that the transgenic plants were originated from independent events of transformation. The positions of prestained molecular markers are indicated on the left in kilodaltons.

Figure 3

Total protein synthesis and glycoprotein accumulation in tunicamycin-treated cell line overexpressing BiP. Wild-type (WT) and 35S-BiPS7 suspension cells were treated with the indicated concentrations of tunicamycin for 12 h and then were labeled for 3 h with [³⁵S]Met and [³⁵S]Cys. Incorporation of radiolabeled amino acids was measured by monitoring trichloroacetic acid (TCA)-precipitable radiolabeled proteins from [³⁵S]Met and [³⁵S]Cys-labeled cell lysates. A, Labeled glycoproteins were affinity purified using concanavalin-A-Sepharose resin and determined by liquid scintillation counting (Beckman Instruments, Fullerton, CA). Relative synthesis of glycoprotein was calculated by normalizing the TCA-precipitable activity (100%) in labeled cell lysates. Values are the mean ± sd from three replicates. B, Protein synthetic rates were calculated as the radioactivity incorporation per microgram of protein. Values are the mean ± sd from three replicates.

Figure 4

Elevated levels of BiP confer water stress tolerance to transgenic plants. Water stress was induced in 6-week-old seedlings (at the same developmental stage) of pBI121-transformed control and transformed 35S-BiPS7 tobacco plants by withholding irrigation for 4 weeks. The stress condition was prolonged until death of control (pBI121) plants (bottom). At the top, 35S-BiPS7 transgenic plants (kanamycin-resistant T₁ generation) were submitted to the same water stress conditions as control plants.

Figure 5

BiP accumulation in transgenic and control leaves during progressive water deficit. Leaf soluble proteins from the transgenic 35S-BiPS7 (A) and pBI121-tranformed control (B) plants grown under water deprivation for 1 week (lane 1), 2 weeks (lane 2), 3 weeks (lane 3), and 4 weeks (lane 4) were fractionated by SDS-PAGE and immunoblotted using an anti-soybean BiP serum. Lane 0 shows the BiP levels in plants before withholding watering.

Figure 6

Antisense expression of BiP genes disrupts the water stress tolerance. Water stress condition was induced in 6-week-old seedlings grown in growth chamber by withholding irrigation for 15 d (from left to right in duplicates): 35S-BiPS3 sense transgenic tobacco plant, pBI121-transformed control plant, and 35S-BiPAS5 antisense plant.

Figure 7

Physiological measurements of transgenic tobacco lines continuously irrigated (black bars) or exposed to 15 d of water deprivation (white bars). On the 15th d of the experiment, the relative water content (D) of well-watered and droughted pBI121-transformed control, 35S-BiPS sense, and 35S-BiPAS antisense transgenic leaves was measured. Photosynthetic rate (A), transpiration rate (B), and stomatal conductance (C) of the third leaf of control, sense, and antisense transgenic plants were measured by the LCA-2 infrared (IR) gas analyzer at 600 μmol m⁻² s⁻¹ irradiance. Each value represents the mean ± sd of five replicates from three independent experiments.

Figure 8

Physiological measurements of sense and antisense transgenic tobacco plants during progressive water deficit. Sense (S), antisense (AS), and PBI121-transformed control (pBI) young seedlings were exposed to the follow water regime: one-half received normal water supply (A, C, and E) and the other half received no irrigation (B, D, and F) during 15 d. Photosynthetic rate (A and B), transpiration rate (C and D), and stomatal conductance (E and F) of the third leaf of pBI121-transformed control, 35S-BiPS, and 35S-BiPAS transgenic plants were measured by the LCA-2 IR gas analyzer at 600 μmol m⁻² s⁻¹ irradiance during the period of the experiment. Each value represents the mean ± sd of five replicates from three independent experiments.