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. 2015 Mar;14(3):686-94.
doi: 10.1074/mcp.M114.042697. Epub 2015 Jan 13.

Proteomic study of microsomal proteins reveals a key role for Arabidopsis annexin 1 in mediating heat stress-induced increase in intracellular calcium levels

Xu Wang  1 , Xiaolong Ma  1 , Hui Wang  1 , Bingjie Li  1 , Greg Clark  2 , Yi Guo  1 , Stan Roux  2 , Daye Sun  1 , Wenqiang Tang  3
Affiliations

Proteomic study of microsomal proteins reveals a key role for Arabidopsis annexin 1 in mediating heat stress-induced increase in intracellular calcium levels

Xu Wang et al. Mol Cell Proteomics. 2015 Mar.

Abstract

To understand the early signaling steps in the response of plant cells to increased environmental temperature, 2-D difference gel electrophoresis was used to study the proteins in microsomes of Arabidopsis seedlings that are regulated early during heat stress. Using mass spectrometry, 19 microsomal proteins that showed an altered expression level within 5 min after heat treatment were identified. Among these proteins, annexin 1 (AtANN1) was one of those up-regulated rapidly after heat-shock treatment. Functional studies show loss-of-function mutants for AtANN1 and its close homolog AtANN2 were more sensitive to heat-shock treatment, whereas plants overexpressing AtANN1 showed more resistance to this treatment. Correspondingly, the heat-induced expression of heat-shock proteins and heat-shock factors is inhibited in ann1/ann2 double mutant, and the heat-activated increase in cytoplasmic calcium concentration ([Ca(2+)]cyt) is greatly impaired in the ann1 mutant and almost undetectable in ann1/ann2 double mutant. Taken together these results suggest that AtANN1 is important in regulating the heat-induced increase in [Ca(2+)]cyt and in the response of Arabidopsis seedlings to heat stress.

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Figures

Fig. 1.
Fig. 1.
The effect of heat shock temperature and duration on the survival of Arabidopsis seedlings. One-week-old Arabidopsis seedlings were heat shock treated at different temperatures for the indicated time period and allowed to recover at 22 °C for 7 days before taking pictures for comparison.
Fig. 2.
Fig. 2.
2D DIGE analysis of heat-regulated microsomal proteins identified AtANN1 as an early heat-responsive protein. A, Microsomal protein from Col-0 seedlings treated with 37 °C for 5 min was labeled with Cy5, and microsomal protein from untreated seedlings was labeled with Cy3. The proteins were then separated using 24 cm, pH 4–7, IPG strips and a 10% SDS-PAGE gel. B, Three-dimensional view of spot 4191 in image A, left image represents Cy3 channel and right image represents Cy5 channel. C, Western analysis of AtANN1 protein accumulation after heat shock. One-week-old seedlings were heat-shock (37 °C) treated for the indicated time, microsomal proteins were extracted and separated by SDS-PAGE. Upper panel shows AtANN1 protein was detected by polyclonal antibodies raised specifically against AtANN1, lower panel shows Ponceau S stained rubisco large subunit as the equal-loading control.
Fig. 3.
Fig. 3.
AtANN1 is essential for plant resistance to heat shock treatment. A, Diagram shows the T-DNA insertion positions for different AtANN1 T-DNA mutants. B, RT-PCR analysis of AtANN1 expression level in different ann1 T-DNA mutants. C, The response of different ann1 T-DNA mutants to heat-shock treatment. Seven-day-old seedlings grown on 1/2 MS plates at 22 °C were shifted to a 45 °C water bath for 13 min and then returned to 22 °C and allowed to recover for 1 week before taking photos. Scale bar represents 1 cm. D, Quantification of survival rate of seedlings shown in C. Data represent the mean value from five independent experiments, with 25 seedlings per experiment. One way ANOVA test was performed. Statistically significant differences are indicated by different lowercase letters (p < 0.05). Error bars represent ±S.D.
Fig. 4.
Fig. 4.
AtANN1 and AtANN2 play redundant roles in regulating plant heat shock responses. A, Diagram shows the T-DNA insertion position for ann2 (SALK_054223) and ann6 (SALK_112492) T-DNA mutants. B, RT-PCR analysis of AtANN2 and AtANN6 expression level in ann2 and ann6 T-DNA mutants. C, The response of ann1–2, ann2, ann6, and ann1–2/ann2 double T-DNA mutant (ann1/2) to heat shock treatment. Seven-day-old seedlings grown on 1/2 MS plates at 22 °C were shifted to a 45 °C water bath for 13 min and then returned to 22 °C and allowed to recover for 1 week before taking photos. Scale bar represents 1 cm.
Fig. 5.
Fig. 5.
The mRNA and protein expression level of several HSFs and HSPs is reduced in ann1–2/ann2 double mutant. A, Semi-quantitative RT-PCR analysis of the mRNA levels of HSF-A7A, HSF-A2, HSF-B1, and HSF-B2A in one-week-old wild type (Col-0) and ann1/ann2 double mutant (ann1/2) seedlings that were HS treated for the indicated time. The RT-PCR products of UBC are shown as the equal-loading control. B, Immunoblot analysis of the protein levels of HSP 17.7, HSP 25.3, HSP 70, and HSP 90 in wild- type and ann1–2/ann2 double mutant. One-week-old seedlings were treated with or without HS for the indicated time, total proteins were separated by SDS-PAGE, blotted onto nitrocellulose membrane and blotted with different HSP specific antibodies. Rubisco large subunit stained by Ponceau S is shown as the equal-loading control.
Fig. 6.
Fig. 6.
Seedlings overexpressing AtANN1 are more resistant to heat shock treatment. A, Upper panel: RT-PCR analysis of the expression level of AtANN1 in overexpressing plants. Lower panel: basal thermotolerance analysis of AtANN1 overexpressing plants. Seven-day-old seedlings grown on 1/2 MS plates at 22 °C were shifted to a 45 °C water bath for 12 min and then returned to 22 °C and allowed to recover for 1 week before taking photos. B, Quantification of survival rate of seedlings shown in A. Data represents the mean value from five independent experiments, with 20 seedlings per experiment. One way ANOVA test was performed. Statistically significant differences are indicated by different lowercase letters (p < 0.05). Error bars represent ±S.D.
Fig. 7.
Fig. 7.
Heat-induced increase in [Ca2+]cyt in seedlings is impaired in Annexin mutants. A, RT-PCR analysis of AtANN1 and AtANN2 expression level in wild type (Col) and ann1–2/ann2 (ann1/2) double T-DNA mutants. B, In vivo reconstitution of aequorin signal in wild type, ann1, and ann1/2 mutants. Error bar represent ±S.E. C, Total calcium abundance in 50 one-week-old seedlings of 35S::Aequorin expressing wild type, ann1 and ann1/2 mutants. One way ANOVA test was performed in B and C. Statistically significant differences are indicated by different lowercase letters (p < 0.05). Error bars represent ±S.D. D, Time-course measurement of aequorin signal in wild type, ann1 and ann1/2 mutants. Seedlings were treated with or without heat shock. Relative aequorin signal is calculated by dividing percentage reconstitution value collected at 37 °C by percentage reconstitution value collected at 22 °C. These experiments have been repeated at least three times, and the trends are similar. Data shown here is from one representative experiment.
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