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The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco.

Valente MA, Faria JA, Soares-Ramos JR, Reis PA, Pinheiro GL, Piovesan ND, Morais AT, Menezes CC, Cano MA, Fietto LG, Loureiro ME, Aragão FJ, Fontes EP - J. Exp. Bot. (2008)

Bottom Line: When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential.It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought.It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, BIOAGRO, Universidade Federal de Viçosa, Avenida PH Rolfs s/n, 36571.000 Viçosa, MG, Brazil.

ABSTRACT
The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

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Ectopic expression of soyBiPD transgene in soybean plants. (A) mRNA abundance of soyBiPD transgene in overexpressing lines under normal growth conditions. Total RNA was isolated from leaves of wild-type (WT) plants and independently transformed soybean lines (35S:BiP-1, 35S:BiP-2, 35S:BiP-3, 35S:BiP-4, and 35S:BiP-5) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers. In the nomenclature of transgenic lines, the first number indicates an independent event of transformation and the second number a different plant in a segregating population. (B) Enhanced levels of BiP in soybean transgenic lines. Equal amount of total proteins (30 μg) extracted from leaves of wild-type plants and soybean transgenic lines (as in A) were separated by SDS-PAGE and immunoblotted with anti-BiP serum. The arrows indicate the positions of BiP and a cross-reacting 28 kDa polypeptide. (C) Immunoblots of whole cell protein extracts (WCE) and microsomal fractions (Mic) of soybean leaves. Whole cell protein extracts from wild-type (lanes 1 and 2), 35S:BiP-2 (lane 3), and 35S-BiP-4 (lane 4) leaves as well as microsomal fractions from wild-type (lanes 5 and 6), 35S:BiP-2 (lane 7), and 35S-BiP-4 (lane 8) leaves were immunoblotted with anti-carboxy BiP serum. (D) Transcript accumulation of soyBiPD transgene in soybean transgenic roots. Total RNA was isolated from roots of wild-type (WT) plants and plants from two independently transformed soybean lines (BiP-2 and BiP-4) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers.
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fig1: Ectopic expression of soyBiPD transgene in soybean plants. (A) mRNA abundance of soyBiPD transgene in overexpressing lines under normal growth conditions. Total RNA was isolated from leaves of wild-type (WT) plants and independently transformed soybean lines (35S:BiP-1, 35S:BiP-2, 35S:BiP-3, 35S:BiP-4, and 35S:BiP-5) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers. In the nomenclature of transgenic lines, the first number indicates an independent event of transformation and the second number a different plant in a segregating population. (B) Enhanced levels of BiP in soybean transgenic lines. Equal amount of total proteins (30 μg) extracted from leaves of wild-type plants and soybean transgenic lines (as in A) were separated by SDS-PAGE and immunoblotted with anti-BiP serum. The arrows indicate the positions of BiP and a cross-reacting 28 kDa polypeptide. (C) Immunoblots of whole cell protein extracts (WCE) and microsomal fractions (Mic) of soybean leaves. Whole cell protein extracts from wild-type (lanes 1 and 2), 35S:BiP-2 (lane 3), and 35S-BiP-4 (lane 4) leaves as well as microsomal fractions from wild-type (lanes 5 and 6), 35S:BiP-2 (lane 7), and 35S-BiP-4 (lane 8) leaves were immunoblotted with anti-carboxy BiP serum. (D) Transcript accumulation of soyBiPD transgene in soybean transgenic roots. Total RNA was isolated from roots of wild-type (WT) plants and plants from two independently transformed soybean lines (BiP-2 and BiP-4) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers.

Mentions: To examine the protective function of BiP against dehydration in soybean, transgenic plants were generated that expressed soyBiPD under the control of the 35S cauliflower mosaic virus promoter. Several independent transgenic lines were established; the expression level of the transgene was analysed by RT-PCR and the accumulation of BiP was monitored in each subsequent generation by immunoblotting. The independently transformed overexpressing (OE) lines constitutively accumulated higher levels of BiPD mRNA (Fig. 1A) and protein (Fig. 1B) than untransformed, wild-type controls. The antibody prepared against purified BiP from soybean seeds cross-reacted with a 28 kDa polypeptide that persisted as a contaminant in the purified BiP fraction used as antigen. To examine whether the ectopically expressed BiP protein was correctly localized in transgenic cells, microsomal fractions were prepared from soybean leaves at the V3 developmental stage and immunoblotted with an anti-BiP serum (Fig. 1C). As expected, BiP was detected in microsomal membrane-enriched fractions from wild-type leaves (lanes 5 and 6) and to a higher extent in microsomal fractions from 35S:BIP-2 and 35S:BiP-4 leaves (lanes 7 and 8). These results indicate that high levels of ectopically expressed BiP were correctly localized in the ER of soybean transgenic leaf cells. Transgene expression, analysed by RT-PCR, was also higher in roots of independently transgenic lines (Fig. 1D).


The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco.

Valente MA, Faria JA, Soares-Ramos JR, Reis PA, Pinheiro GL, Piovesan ND, Morais AT, Menezes CC, Cano MA, Fietto LG, Loureiro ME, Aragão FJ, Fontes EP - J. Exp. Bot. (2008)

Ectopic expression of soyBiPD transgene in soybean plants. (A) mRNA abundance of soyBiPD transgene in overexpressing lines under normal growth conditions. Total RNA was isolated from leaves of wild-type (WT) plants and independently transformed soybean lines (35S:BiP-1, 35S:BiP-2, 35S:BiP-3, 35S:BiP-4, and 35S:BiP-5) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers. In the nomenclature of transgenic lines, the first number indicates an independent event of transformation and the second number a different plant in a segregating population. (B) Enhanced levels of BiP in soybean transgenic lines. Equal amount of total proteins (30 μg) extracted from leaves of wild-type plants and soybean transgenic lines (as in A) were separated by SDS-PAGE and immunoblotted with anti-BiP serum. The arrows indicate the positions of BiP and a cross-reacting 28 kDa polypeptide. (C) Immunoblots of whole cell protein extracts (WCE) and microsomal fractions (Mic) of soybean leaves. Whole cell protein extracts from wild-type (lanes 1 and 2), 35S:BiP-2 (lane 3), and 35S-BiP-4 (lane 4) leaves as well as microsomal fractions from wild-type (lanes 5 and 6), 35S:BiP-2 (lane 7), and 35S-BiP-4 (lane 8) leaves were immunoblotted with anti-carboxy BiP serum. (D) Transcript accumulation of soyBiPD transgene in soybean transgenic roots. Total RNA was isolated from roots of wild-type (WT) plants and plants from two independently transformed soybean lines (BiP-2 and BiP-4) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2651463&req=5

fig1: Ectopic expression of soyBiPD transgene in soybean plants. (A) mRNA abundance of soyBiPD transgene in overexpressing lines under normal growth conditions. Total RNA was isolated from leaves of wild-type (WT) plants and independently transformed soybean lines (35S:BiP-1, 35S:BiP-2, 35S:BiP-3, 35S:BiP-4, and 35S:BiP-5) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers. In the nomenclature of transgenic lines, the first number indicates an independent event of transformation and the second number a different plant in a segregating population. (B) Enhanced levels of BiP in soybean transgenic lines. Equal amount of total proteins (30 μg) extracted from leaves of wild-type plants and soybean transgenic lines (as in A) were separated by SDS-PAGE and immunoblotted with anti-BiP serum. The arrows indicate the positions of BiP and a cross-reacting 28 kDa polypeptide. (C) Immunoblots of whole cell protein extracts (WCE) and microsomal fractions (Mic) of soybean leaves. Whole cell protein extracts from wild-type (lanes 1 and 2), 35S:BiP-2 (lane 3), and 35S-BiP-4 (lane 4) leaves as well as microsomal fractions from wild-type (lanes 5 and 6), 35S:BiP-2 (lane 7), and 35S-BiP-4 (lane 8) leaves were immunoblotted with anti-carboxy BiP serum. (D) Transcript accumulation of soyBiPD transgene in soybean transgenic roots. Total RNA was isolated from roots of wild-type (WT) plants and plants from two independently transformed soybean lines (BiP-2 and BiP-4) and BiP transgene transcript levels were quantified by real-time PCR, using transgene-specific primers.
Mentions: To examine the protective function of BiP against dehydration in soybean, transgenic plants were generated that expressed soyBiPD under the control of the 35S cauliflower mosaic virus promoter. Several independent transgenic lines were established; the expression level of the transgene was analysed by RT-PCR and the accumulation of BiP was monitored in each subsequent generation by immunoblotting. The independently transformed overexpressing (OE) lines constitutively accumulated higher levels of BiPD mRNA (Fig. 1A) and protein (Fig. 1B) than untransformed, wild-type controls. The antibody prepared against purified BiP from soybean seeds cross-reacted with a 28 kDa polypeptide that persisted as a contaminant in the purified BiP fraction used as antigen. To examine whether the ectopically expressed BiP protein was correctly localized in transgenic cells, microsomal fractions were prepared from soybean leaves at the V3 developmental stage and immunoblotted with an anti-BiP serum (Fig. 1C). As expected, BiP was detected in microsomal membrane-enriched fractions from wild-type leaves (lanes 5 and 6) and to a higher extent in microsomal fractions from 35S:BIP-2 and 35S:BiP-4 leaves (lanes 7 and 8). These results indicate that high levels of ectopically expressed BiP were correctly localized in the ER of soybean transgenic leaf cells. Transgene expression, analysed by RT-PCR, was also higher in roots of independently transgenic lines (Fig. 1D).

Bottom Line: When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential.It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought.It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, BIOAGRO, Universidade Federal de Viçosa, Avenida PH Rolfs s/n, 36571.000 Viçosa, MG, Brazil.

ABSTRACT
The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought.

Show MeSH
Related in: MedlinePlus