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Developmental and heat stress-regulated expression of HsfA2 and small heat shock proteins in tomato anthers.

Giorno F, Wolters-Arts M, Grillo S, Scharf KD, Vriezen WH, Mariani C - J. Exp. Bot. (2009)

Bottom Line: A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR.The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis.These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Cell Biology, IWWR Institute, Radboud University Nijmegen, Heyendaalseweg 135, Nijmegen, The Netherlands.

ABSTRACT
The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

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Expression analyses of HsfA2 and Hsp17-CII during anther development. (A–C) Relative mRNA levels of HsfA2 (A), Hsp17.4-CII (B), and Hsp17.6-CII (C) in different sized anthers under CT conditions (26 °C / 19 °C, day/night). Expression data were normalized using LeEF1 and 18S rRNA as housekeeping genes. The mRNA levels of the target genes in 4, 6, and 8 mm anthers are relative to that from 2 mm anthers (value 1). The experiment shown here is one of two biological replicates, which showed a comparable expression pattern. Each graph represents the data of two technical repeats. (D) Immunoblotting analyses of the proteins isolated from the same samples as in A, B, and C using specific antisera against HsfA2 and Hsp17-CII. Ponceau staining of total protein was used as control for equal loading.
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fig3: Expression analyses of HsfA2 and Hsp17-CII during anther development. (A–C) Relative mRNA levels of HsfA2 (A), Hsp17.4-CII (B), and Hsp17.6-CII (C) in different sized anthers under CT conditions (26 °C / 19 °C, day/night). Expression data were normalized using LeEF1 and 18S rRNA as housekeeping genes. The mRNA levels of the target genes in 4, 6, and 8 mm anthers are relative to that from 2 mm anthers (value 1). The experiment shown here is one of two biological replicates, which showed a comparable expression pattern. Each graph represents the data of two technical repeats. (D) Immunoblotting analyses of the proteins isolated from the same samples as in A, B, and C using specific antisera against HsfA2 and Hsp17-CII. Ponceau staining of total protein was used as control for equal loading.

Mentions: The observation that 2 mm anthers from CT plants had the highest expression of HsfA2 and Hsp17.4-CII led to a more detailed investigation of transcript levels and protein accumulation under CT conditions. As shown in Fig. 3A, HsfA2 expression in 2 mm anthers from CT plants was higher than that in older ones. Correspondingly, the amount of the protein was higher at the 2 mm and 4 mm anther stages, subsequently decreasing in 6 mm and 8 mm anthers (Fig. 3D). The Hsp17.4-CII gene was also expressed during anther development (Fig. 3B), particularly at the younger stages, when the accumulation of Hsp17-CII proteins was also observed (Fig. 3D). However, because tomato small Hsps, such as Hsp17.4-CII and Hsp17.6-CII, belong to the same subfamily and differ only by a few amino acid residues, it was impossible to discriminate them by the antibody used in this study (Port et al., 2004). This led to an analysis of the Hsp17.6-CII transcript profiles in order to distinguish the Hsp17.4-CII and Hsp17.6-CII mRNAs that are translated into the respective proteins. As observed for Hsp17.4-CII, the Hsp17.6-CII gene was also expressed during anther development under CT conditions, particularly in 2 mm anthers (Fig. 3C), indicating that the Hsp17.4-CII and Hsp17.6-CII proteins may be detected together with the Hsp17-CII antibody.


Developmental and heat stress-regulated expression of HsfA2 and small heat shock proteins in tomato anthers.

Giorno F, Wolters-Arts M, Grillo S, Scharf KD, Vriezen WH, Mariani C - J. Exp. Bot. (2009)

Expression analyses of HsfA2 and Hsp17-CII during anther development. (A–C) Relative mRNA levels of HsfA2 (A), Hsp17.4-CII (B), and Hsp17.6-CII (C) in different sized anthers under CT conditions (26 °C / 19 °C, day/night). Expression data were normalized using LeEF1 and 18S rRNA as housekeeping genes. The mRNA levels of the target genes in 4, 6, and 8 mm anthers are relative to that from 2 mm anthers (value 1). The experiment shown here is one of two biological replicates, which showed a comparable expression pattern. Each graph represents the data of two technical repeats. (D) Immunoblotting analyses of the proteins isolated from the same samples as in A, B, and C using specific antisera against HsfA2 and Hsp17-CII. Ponceau staining of total protein was used as control for equal loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803211&req=5

fig3: Expression analyses of HsfA2 and Hsp17-CII during anther development. (A–C) Relative mRNA levels of HsfA2 (A), Hsp17.4-CII (B), and Hsp17.6-CII (C) in different sized anthers under CT conditions (26 °C / 19 °C, day/night). Expression data were normalized using LeEF1 and 18S rRNA as housekeeping genes. The mRNA levels of the target genes in 4, 6, and 8 mm anthers are relative to that from 2 mm anthers (value 1). The experiment shown here is one of two biological replicates, which showed a comparable expression pattern. Each graph represents the data of two technical repeats. (D) Immunoblotting analyses of the proteins isolated from the same samples as in A, B, and C using specific antisera against HsfA2 and Hsp17-CII. Ponceau staining of total protein was used as control for equal loading.
Mentions: The observation that 2 mm anthers from CT plants had the highest expression of HsfA2 and Hsp17.4-CII led to a more detailed investigation of transcript levels and protein accumulation under CT conditions. As shown in Fig. 3A, HsfA2 expression in 2 mm anthers from CT plants was higher than that in older ones. Correspondingly, the amount of the protein was higher at the 2 mm and 4 mm anther stages, subsequently decreasing in 6 mm and 8 mm anthers (Fig. 3D). The Hsp17.4-CII gene was also expressed during anther development (Fig. 3B), particularly at the younger stages, when the accumulation of Hsp17-CII proteins was also observed (Fig. 3D). However, because tomato small Hsps, such as Hsp17.4-CII and Hsp17.6-CII, belong to the same subfamily and differ only by a few amino acid residues, it was impossible to discriminate them by the antibody used in this study (Port et al., 2004). This led to an analysis of the Hsp17.6-CII transcript profiles in order to distinguish the Hsp17.4-CII and Hsp17.6-CII mRNAs that are translated into the respective proteins. As observed for Hsp17.4-CII, the Hsp17.6-CII gene was also expressed during anther development under CT conditions, particularly in 2 mm anthers (Fig. 3C), indicating that the Hsp17.4-CII and Hsp17.6-CII proteins may be detected together with the Hsp17-CII antibody.

Bottom Line: A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR.The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis.These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Cell Biology, IWWR Institute, Radboud University Nijmegen, Heyendaalseweg 135, Nijmegen, The Netherlands.

ABSTRACT
The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

Show MeSH
Related in: MedlinePlus