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• W2157017347 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 ([Ca2+]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 [Ca2+]cyt and in the response of Arabidopsis seedlings to heat stress. 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 ([Ca2+]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 [Ca2+]cyt and in the response of Arabidopsis seedlings to heat stress. Temperatures above the optimum are sensed as heat stress (HS) 1The abbreviations used are:CHAPS3-Cholamidopropyl dimethylammonio]-1-propanesulfonateHSheat stressHSFsheat shock transcription factorsHSPsheat shock proteinsAtANN1Arabidopsis annexin 1CDPKscalcium dependent protein kinasesHSRheat-shock responsesCol-0Columbia-0CNGCcyclic nucleotide-gated channelsMS mediumMurashige and Skoog medium[Ca2+]cytcytoplasmic calcium concentrationPLCphospholipase CROSReactive oxygen species. 1The abbreviations used are:CHAPS3-Cholamidopropyl dimethylammonio]-1-propanesulfonateHSheat stressHSFsheat shock transcription factorsHSPsheat shock proteinsAtANN1Arabidopsis annexin 1CDPKscalcium dependent protein kinasesHSRheat-shock responsesCol-0Columbia-0CNGCcyclic nucleotide-gated channelsMS mediumMurashige and Skoog medium[Ca2+]cytcytoplasmic calcium concentrationPLCphospholipase CROSReactive oxygen species. by all living organisms. When exposed to high temperature, plants have sophisticated mechanisms to maintain cellular homeostasis and minimize cell damage. Heat shock transcription factors (HSFs) mediate gene expression changes leading to the production of heat shock proteins (HSPs) that confer thermotolerance to plants (1Baniwal S.K. Bharti K. Chan K.Y. Fauth M. Ganguli A. Kotak S. Mishra S.K. Nover L. Port M. Scharf K.D. Tripp J. Weber C. Zielinski D. Con Koskull-Doring P. Heat stress response in plants, a complex game with chaperones and more than twenty heat stress transcription factors.J. Biosci. 2004; 29: 471-487Crossref PubMed Scopus (408) Google Scholar, 2Nover L. Bharti K. Doring P. Mishra S.K. Ganguli A. Scharf K.D. Arabidopsis and the heat stress transcription factor world, how many heat stress transcription factors do we need?.Cell Stress Chaperones. 2001; 6: 177-189Crossref PubMed Google Scholar, 3Pirkkala L. Nykanen P. Sistonen L. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond.FASEB J. 2001; 15: 1118-1131Crossref PubMed Scopus (820) Google Scholar). In recent years, there has been considerable progress in identifying the signaling steps that enable plants to respond to heat stress at the molecular level. These include: activation of bZIP transcription factors by heat-induced proteolytic cleavage (4Deng Y. Humbert S. Liu J.X. Srivastava R. Rothstein S. Howell S.H. Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 7247-7252Crossref PubMed Scopus (317) Google Scholar); inhibition of transgene-induced post-transcriptional gene silencing (PTGS) by SUPPRESSOR OF GENE SILENCING 3 (SGS3) (5Zhong S.H. Liu J.Z. Jin H. Lin L. Li Q. Chen Y. Yuan Y.Z. Wang Z.-Y. Huang H. Qi Y.J. Chen X.Y. Vaucheret H. Chory J. Li J.M. He Z.H. Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 9171-9176Crossref PubMed Scopus (79) Google Scholar); accumulation of reactive oxygen species (ROS) by RBOHD activation (6Mittler R. Finka A. Goloubinoff P. How do plants feel the heat?.Trends Biochem. Sci. 2012; 37: 118-125Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar); and reduced H2A.Z occupancy in nucleosomes of heat-regulated genes, which would be expected to stimulate transcription of these genes (7Kumar S.V. Wgge P.A. H2A.Z containing nucleosomes mediate the thermosensory response in Arabidopsis.Cell. 2010; 140: 136-147Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar). 3-Cholamidopropyl dimethylammonio]-1-propanesulfonate heat stress heat shock transcription factors heat shock proteins Arabidopsis annexin 1 calcium dependent protein kinases heat-shock responses Columbia-0 cyclic nucleotide-gated channels Murashige and Skoog medium cytoplasmic calcium concentration phospholipase C Reactive oxygen species. 3-Cholamidopropyl dimethylammonio]-1-propanesulfonate heat stress heat shock transcription factors heat shock proteins Arabidopsis annexin 1 calcium dependent protein kinases heat-shock responses Columbia-0 cyclic nucleotide-gated channels Murashige and Skoog medium cytoplasmic calcium concentration phospholipase C Reactive oxygen species. Many reports also provide evidence that membrane-localized calcium channels can act as sensors for increased temperature in plant cells (8Finka A. Cuendet A.F.H. Maathuis F.J.M. Saidi Y. Goloubinoff P. Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.Plant Cell. 2012; 24: 3333-3348Crossref PubMed Scopus (215) Google Scholar, 9Gao F. Han X.W. Wu J.H. Zheng S.Z. Shang Z.L. Sun D.Y. Zhou R.G. Li B. A heat activated calcium permeable channel, Arabidopsis cyclic nucleotide-gated ion channel 6, is involved in heat shock responses.Plant J. 2012; 70: 1056-1069Crossref PubMed Scopus (132) Google Scholar). The increase in cytosolic calcium concentration ([Ca2+]cyt) caused by calcium influx from the extracellular matrix is an early critical step in the HS signal transduction pathway (10Saidi Y. Finka A. Goloubinoff P. Heat perception and signaling in plants: a tortuous path to thermotolerance.New Phytol. 2010; 190: 556-565Crossref PubMed Scopus (187) Google Scholar). In responding to this calcium influx, calcium-binding proteins such as calmodulin and calcium dependent protein kinases (CDPKs) are activated. Calmodulin binds to a calmodulin-binding protein kinase, which in turn, activates heat-regulated changes in gene expression by phosphorylating HSF (11Liu H.T. Li B. Shang Z.L. Li X.Z. Mu R.L. Sun D.Y. Zhou R.G. Calmodulin is involved in heat shock signal transduction in wheat.Plant Physiol. 2003; 132: 1186-1195Crossref PubMed Scopus (164) Google Scholar, 12Liu H.T. Gao F. Li G.L. Han J.L. Liu D.L. Sun D.Y. Zhou R.G. The calmodulin-binding protein kinase 3 is part of heat shock signal transduction in Arabidopsis thaliana.Plant J. 2008; 55: 760-773Crossref PubMed Scopus (171) Google Scholar, 13Zhang W. Zhou R.G. Gao Y.J. Zheng S.Z. Xu P. Zhang S.Q. Sun D.Y. Molecular and Genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis.Plant Physiol. 2009; 149: 1773-1784Crossref PubMed Scopus (183) Google Scholar). CDPKs, on the other hand, might regulate plant heat-shock responses by activating multiple mitogen-activated protein kinases (14Sangwan V. Orvar B.L. Beyerly J. Hirt H. Dhindsa R.S. Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways.Plant J. 2002; 31: 629-638Crossref PubMed Scopus (288) Google Scholar). Despite considerable progress in studying the mechanisms of heat-shock signal transduction, our understanding of the heat signal perception and early signaling transduction mechanism is still very limited. Therefore, identifying novel proteins that regulate early heat-shock responses should help decoding high-temperature sensing and responding mechanism in planta. Along with advances in genome sequencing, new proteomic technologies have now emerged as an important tool in studying signal transduction mechanisms (15Tang W.Q. Deng Z.P. Wang Z.Y. Protomics shed light on the brassinosteroid signaling mechanisms.Curr. Opin. Plant Biol. 2010; 13: 27-33Crossref PubMed Scopus (58) Google Scholar). Compared with traditional genetic studies, which rely on phenotypes caused by altering gene activity, proteomics not only provides real-time monitoring of abundance changes of certain proteins during cell and tissue responses to stimuli, but also reveals their mechanisms of function and regulation, such as post-translational modifications and protein–protein interaction. In this study, we used the two-dimensional difference gel electrophoresis (2-D DIGE) technology to find microsome-associated proteins whose level changes rapidly after heat-shock, and identified a set of membrane proteins that change abundance in response to a five-minute heat treatment. Among these proteins was annexin 1 (AtANN1), and here we demonstrate that this protein plays a key role in plant heat-stress responses by mediating a heat-induced increase in [Ca2+]cyt. Arabidopsis thaliana seeds were surface sterilized using 75% (v/v) ethanol and sowed in glass plates containing 25 ml growth medium containing half-strength Murashige and Skoog (1/2 MS) salt, 1.5% (w/v) sucrose, and 0.8% (w/v) agar. Plates were stratified at 4 °C in darkness for 3 days. Seedlings were allowed to grow at 22 °C under long-day conditions (16 h light/8 h dark) for 1 week. For heat shock treatment, the glass plates were wrapped with general kitchen plastic wrap and submerged in a water bath at the indicated temperature for different periods. The seedlings were returned to 22 °C under long-day to recover for 1 week before taking pictures for analysis. All the data showed in this study has been performed at least three times with similar results. Representative data from one repetition are shown in the figures. Because we were interested in finding heat shock-regulated proteins whose level changed rapidly, we used a water bath for our heat shock proteomic studies to make sure all the seedlings were instantly subjected to similar temperature at same time. To prepare the seedlings for 2D-DIGE protein sample extraction, Arabidopsis seedlings were grown in liquid medium containing 1/2 MS salt and 1.5% (w/v) sucrose under long day at 22 °C for 1 week. To avoid a nutrient shock effect, half of the old medium was transferred into a new container and incubated at 37 °C in the water bath, whereas the other half of the medium was kept at 22 °C with the seedlings. When the temperature of the water bath medium reached 37 °C, half of the seedlings were transferred into the medium to initiate the heat shock treatment. After heat shock treatment for 5 min, seedlings were harvested by filtering through a nylon mesh, quick tap-dried with tissue paper and frozen in liquid nitrogen. Microsomal proteins were extracted as described previously (16Tang W.Q. Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis.Methods Mol. Biol. 2012; 876: 67-82Crossref PubMed Scopus (11) Google Scholar), and dissolved in DIGE buffer (7 m urea, 2 m thiourea, 4% CHAPS) at a concentration around 5–10 μg/μl. Protein for 2D-DIGE analysis was labeled with CyDyeTM DIGE fluor minimal labeling kit (GE Healthcare). In brief, 10 μl microsomal protein from control or heat treated sample was mixed with 0.25 μl of Tris-HCl (1.5 m, pH 8.8) and 0.5 μl of 100 nm Cy3 or Cy5, respectively, and incubated in darkness at 4 °C for 2 h. The reaction was terminated by adding 1 μl 10 mm lysine and incubating with the protein on ice for 10 min. The Cy3- and Cy5- labeled pair of control and heat treated samples were combined, and to the mix was added 9 μl of 1 m DTT, 4.5 μl of IPG buffer, pH 4–7. One hundred-micrograms of unlabeled protein each from control and heat-treated samples was then added to make the total protein amount in the mixture around 300 μg. The DIGE buffer was used to adjust the final volume to 450 μl for separation by isoelectric focusing (IEF) using 24 cm Immobiline Dry Strips, pH 4–7. Two-dimensional electrophoresis was performed according to (16Tang W.Q. Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis.Methods Mol. Biol. 2012; 876: 67-82Crossref PubMed Scopus (11) Google Scholar). DIGE images were acquired using a Typhoon trio scanner (GE healthcare). At least six biological repeats were performed, and labeling of two pairs of biological repeat samples was swapped to avoid identifying proteins that are preferentially labeled by a specific CyDye. Spots that showed consistent heat shock regulated changes in at least five biological repeat samples were picked using a robotic spot picker (GE healthcare). Protein in-gel digestion was performed as described previously (17Tang W.Q. Deng Z.P. Oses-Prieto J. Suzuki N. Zhu S.W. Zhang X. Burlingame A.L. Wang Z.Y. Proteomics studies of brassinosteroid signal transduction using prefractionation and two dimensional DIGE.Mol. Cell. Proteomics. 2008; 7: 728-738Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Extracted peptides in 0.1% formic acid were separated using a 100 μm ×150 mm C18 reverse phase column (Thermo Fisher Scientific) at a flow rate of 350 nl/min, and eluted using a linear acetonitrile (ACN) gradient (0–45%) with 0.1% formic acid over 60 min using a nano-LC system (Thermo Fisher Scientific). Eluted peptides were electrosprayed directly into a LTQ-XL linear ion trap mass spectrometer (Thermo Fisher Scientific) using an uncoated 15 μm inner diameter spraying needle with 2.1 kV electrospray voltage at 220 °C. Peptides were analyzed in positive ion mode with m/z range between 400 and 2000. Charged peaks over 1000 counts were selected for collision induced dissociation (CID) with 35% normalized collision energy and activation Q value was set to 0.25. The dynamic exclusion activation time was set to 30 s to prevent same m/z ions from being selected after its acquisition. CID product ions were analyzed on the linear ion trap in centroid mode. Complete LC-MS/MS peak lists were searched against database generated from Arabidopsis subset of the NCBInr database (date 12/12/2012, 257,035 entries searched), using the SEQUEST search algorithm, which is part of the BioWorks 3.3.1 data analysis software (Thermo Fisher Scientific). The peptide mass range was set to 400–5000 amu. The precursor ions' tolerance and fragment ions' tolerance were set to 2.00 amu and 1.00 amu, respectively. Enzymatic digestion was specified as trypsin, with a maximum of two missed cleavages allowed. Cysteine carbamidomethylation was allowed as fixed modifications, and methionine oxidation were allowed as variable modification. Search result option filter was set as the following: Delta CN ≥ 0.1; Xcorr (±1, 2, 3) = 1.75, 2.5, 3.0; peptide probability≤0.01. T-DNA insertional mutant for AtANN1 (SALK_132169, SAIK_414_C01, WiscDsLox477–480P11), AtANN2 (SALK_054223) and AtANN6 (SALK_112492) were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org), and are all in Columbia (Col-0) background. The identified ann1–2 mutant was used to cross with ann2 mutant. F3 segregated ann1–2/ann2 double homozygous mutant were used for heat stress tolerance analysis. The gene specific primers used for genotyping the mutants were: 5′-gcctgcttcagcttttgtatg-3′ (left) and 5′-aacgctaccgacacaacattc-3′ (right) for SALK_132169; 5′-tgctccttctgatgatgctg-3′ (left) and 5′-ccaataaagcatcacgctca-3′ (right) for SAIL_414_C01; 5′-tggactcttgatccaccaga-3′ (left) and 5′-gagcaacaagcatgtcctca-3′ (right) for SALK_054223; 5′-ggcgtctctcaaaattccag-3′ (left) and 5′-caccagaaagctctccgtct-3′ (right) for SALK_112492. The transcript abundance of AtANN1 and AtANN2 in wild type and mutant seedlings was determined by semi-quantitative Reverse transcription PCR using 5′-atggcgactcttaaggtttc-3′ (left) and 5′-agcatcatcttcaccgagaag-3′ (right) for AtANN1 and gene specific genotyping primer set for SALK_054223 and SALK_112492. Full-length CDS of AtANN1 were amplified by PCR, cloned into pENTR/SD/D-TOPO vector (Invitrogen), and subcloned into Gateway compatible binary vector pEarleyGate 100 by LR clonase (Invitrogen). The construct was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and transformed into Arabidopsis (Col-0 ecotype) by the floral dipping method. Transgenic plants were selected by spraying plants with 5 μg/ml Basta (Finale). All transgenic lines used in this study are single insertional homozygous T3 lines. Wild type and ann1–2/ann2 double mutant seedlings were grown in the same glass plates containing 15 ml growth medium (1/2 MS salt, 1.5% sucrose, and 0.8% agar) for 1 week at 22 °C under long-day condition. For heat shock treatment, the plates were placed into a growth chamber with temperature set at 37 °C. Seedlings were harvested at different time intervals and frozen in liquid nitrogen. For RT-PCR analysis of the expression level of various HSFs transcript, total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using ExScriptTM RT reagent kit (Takara) according to manufacturer's instruction. Primer pairs used for RT-PCR analysis were: 5′-atgatgaacccgtttctcccggaag-3′ and 5′-ggaggtggaagccaaactctcatcac-3′ for HSF A7A; 5′-atggaagaactgaaagtggaaat-3′ and 5′-aggttccgaaccaagaaaacccatt-3′ for HSF A2; 5′-atgacggctgtgacggcggcgcaaag-3′ and 5′-gttgcagactttgctgcttttccac-3′ for HSF B1; 5′-acagatttcgctaaagatttgctt-3′ and 5′-cgcttcttcttcttcttcaatctc-3′ for HSF B2A. For protein abundance analysis of HSPs, frozen tissues were ground into fine powder in liquid nitrogen, boiled with 2× SDS sampling buffer, loaded onto SDS-PAGE and transferred onto nitrocellulose membrane. Membrane was probed with different antibodies specifically against various HSPs (Agrisera, Vännäs, Sweden) and detected with SuperSignal West Dura Chemiluminescence System (Pierce). Aequorin luminescence measurement was performed according to (18Knight H. Trewavas A.J. Knight M.R. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation.Plant Cell. 1996; 8: 489-503Crossref PubMed Scopus (722) Google Scholar), with slight modifications. Transgenic Arabidopsis seedlings expressing cytosolic 35S::Aequorin were grown in 1/2 MS salt containing medium for 10 days under long day at 22 °C. Seedlings (n ≥ 15 per treatment) were transferred to a 96-well plate and incubated in dark with a calcium-measurement buffer (0.1 mm KCl, 1 mm CaCl2, 10 mm MES, 5 μm coelenterazine-h) at 22 °C for 6 h. The seedlings were quick rinsed two times with the calcium-measurement buffer, 100 μl 22 °C (control) or 37 °C (heat shock) calcium measurement buffer was quickly added to each well, and the plate was loaded into a microplate luminometer (Centro LB 960, Berthold, Oak Ridge, TN) that is at room temperature (control) or has been prewarmed to 37 °C (heat shock). Aequorin luminescence signal in each well was recorded for 0.5 s every 30 s. After 45 counts, 100 μl reconstitution solution (2 m CaCl2, 20% ethanol) was injected and luminescence signal in each well was recorded for 10 more cycles, 0.5 s each. Percentage reconstitution value was calculated by dividing the luminescence signal recorded at each time point by the highest reconstitution luminescence value recorded in each well, respectively, for wild type, ann1 and ann1/2 mutants. Relative aequorin signal is calculated by dividing percentage reconstitution value collected at 37 °C by percentage reconstitution value collected at 22 °C. Fifty seven-day-old Arabidopsis seedlings grown in 1/2 MS medium supplied with 1% sucrose were grouped as one sample and oven dried at 65 °C overnight. After recording the dry weights, the dried plants were digested with 5 ml HNO3 at 100 °C for 4 h. The digested mixture was transferred to a volumetric flask, and diluted precisely to 100 ml with double distilled water. Total plant Ca2+ concentration was determined with a flame atomic absorption spectrophotomer (Model AA-240Z, Agilent Technologies, Santa Clara, CA). Increased environmental temperature triggers heat-shock responses (HSR) in plants; however, if the temperature rises above a certain limit, or the duration of the high temperature is too long, plants will not be able to survive. To study the HS response of plant cells, the treatment used should be strong enough to stimulate the HSR, but at the same time mild enough not to kill the plant. In order to find a proper heat-shock treatment condition for our proteomic studies, plates containing one-week-old Arabidopsis seedlings were treated at a range of temperatures and treatment durations, and post-treatment survival rates were calculated after the seedlings recovered at 22 °C for 1 week. Treatment of Arabidopsis seedlings in a 42 °C water bath up to 10 min did not induce irreversible damage to seedlings, whereas treatment with a 42 °C water bath for 30 min killed almost all of the seedlings. In comparison, seedlings treated with a 37 °C water bath for 30 min survived (Fig. 1). As 37 °C has been previously shown to induce HSR (9Gao F. Han X.W. Wu J.H. Zheng S.Z. Shang Z.L. Sun D.Y. Zhou R.G. Li B. A heat activated calcium permeable channel, Arabidopsis cyclic nucleotide-gated ion channel 6, is involved in heat shock responses.Plant J. 2012; 70: 1056-1069Crossref PubMed Scopus (132) Google Scholar, 11Liu H.T. Li B. Shang Z.L. Li X.Z. Mu R.L. Sun D.Y. Zhou R.G. Calmodulin is involved in heat shock signal transduction in wheat.Plant Physiol. 2003; 132: 1186-1195Crossref PubMed Scopus (164) Google Scholar, 12Liu H.T. Gao F. Li G.L. Han J.L. Liu D.L. Sun D.Y. Zhou R.G. The calmodulin-binding protein kinase 3 is part of heat shock signal transduction in Arabidopsis thaliana.Plant J. 2008; 55: 760-773Crossref PubMed Scopus (171) Google Scholar), a 37 °C water bath was used as our treatment temperature during the following proteomic studies. Heat shock stimulates a rapid increase of the [Ca2+]cyt. This increase can be seen as early as 1 min after heat treatment, and reaches the maximum around 5 mins (11Liu H.T. Li B. Shang Z.L. Li X.Z. Mu R.L. Sun D.Y. Zhou R.G. Calmodulin is involved in heat shock signal transduction in wheat.Plant Physiol. 2003; 132: 1186-1195Crossref PubMed Scopus (164) Google Scholar). In order to study the early cellular responses stimulated by HS, we exposed one-week-old Arabidopsis seedlings to 37 °C for 5 mins and harvested them for 2D-DIGE analysis. Protein samples from control and treated seedlings were analyzed in at least six biological-repeat experiments. In average, over 1500 protein spots were detected in each gel (Fig. 2A). Examination of the DIGE images identified over 49 spots that showed consistent abundance changes induced by heat shock. Surprisingly, the abundance of most spots was increased by heat treatment, and only a few showed decreased levels. These spots of interest were picked, digested in gel by trypsin and analyzed by LC-MS/MS. A total of 27 heat-shock-regulated protein spots, which represented 19 unique proteins, were successfully identified (Table I). Functional classification showed these proteins are involved in processes such as hormone biosynthesis, protein translation, calcium signaling, cellular metabolism, and protein folding (chaperone).Table IEarly heat-regulated microsomal proteins identified by mass spectrometry. The heat regulated abundance changes were observed from at least five biological repeats. For MS/MS identification, number of unique peptides, sequence coverage of the identified proteins and the best matched scores are listedSpotsAccession numberProtein nameUnique peptidesCoverage (%)Abundance change by HSScoreHormone biosynthesis2586At2g46370Jasmonate-amido synthetase36.26up30.193984At1g62380ACC oxidase 2832.81up70.284095At3g44310Nitrilase 1937.28down100.284125At3g44310Nitrilase 11250down130.294285At3g44310Nitrilase 1625.72down70.27Protein translation3384At1g57720Translation elongation factor EF1B512.35up50.233394At1g57720Translation elongation factor EF1B415.5down50.243421At1g57720Translation elongation factor EF1B1026.63up170.293415At3g13920Eukaryotic translation initiation factor 4A-1728.16up100.313424At3g13920Eukaryotic translation initiation factor 4A-1834.22down120.323804At4g20360GTP binding Elongation factor Tu family protein927.73up80.31Calcium signaling4181At1g35720Annexin 126.308up40.244191At1g35720Annexin 1515.77up70.28Protein folding2371At5g42020Luminal binding protein (BIP2)34.8up30.192407At5g42020Luminal binding protein (BIP2)611.98up50.246119At3g62030Cyclophilin 20–3 (CYP20–3)524.52up50.25Metabolism1969At1g08520CHLD subunit of the Mg-chelatase enzyme1625.58up160.233874At1g73110Nucleoside triphosphate hydrolase1440.05up170.274097At2g058305-methylthioribose-1-phosphate isomerase725.67up80.244113At5g15650AtRGP2411.67up40.234809At2g34470Urease accessory protein G (UREG)526.55up50.315092At4g30530Gamma-glutamyl peptidase317.60up30.265128At3g02780Isopentenyl diphosphate isomerase 2519.01up50.285116At3g11930Adenine nucleotide alpha hydrolases like211.5up20.19Others2060At1g33790Jacalin lectin family protein411.69up40.172062At1g33790Jacalin lectin family protein1436.85up120.314734At4g39090Cysteine proteinases39.24down30.22 Open table in a new tab Of all the microsomal proteins identified in Table I, we chose to further study AtANN1 (Fig. 2B), because of prior evidence showing that it could play a major role in mediating plant stress responses. Using polyclonal antibody raised specific against a 31 amino acid sequence from AtANN1 (amino acids 200–231) (19Clark G.B. Lee D. Dauwalder M. Roux S.J. Immunolocalization and histochemical evidence for the association of two different Arabidopsis annexins with secretion during early seedling growth and development.Planta. 2005; 220: 621-631Crossref PubMed Scopus (50) Google Scholar), we confirmed the 2D-DIGE result in which AtANN1 protein levels increased in the microsomal fraction after 5 mins of HS (Fig. 2C). We then isolated Transfer DNA (T-DNA) insertion mutants of AtANN1 gene to investigate if AtANN1 plays a significant role in HS responses in Arabidopsis. We obtained three insertion mutants for AtANN1, and these alleles were named as ann1–1 (SalK_132169), ann1–2 (Sail_414_C01), and ann1–3 (WiscDsLox477–480P11; Fig. 3A). The T-DNA insertion sites in these alleles were located at the 5′ UTR of the AtANN1 gene (ann1–1) or within different positions of its second and third exon (ann1–2 and ann1–3), respectively (Fig. 3A). Reverse transcription PCR (RT-PCR) analysis of these T-DNA mutants showed that the expression of AtANN1 in ann1–2 and ann1–3 is not detectable, whereas the expression of AtANN1 in ann1–1 was not affected (Fig. 3B). We then compared the basal thermotolerance of these T-DNA insertional mutants with wild-type seedlings. One-week-old seedlings were subjected to 45 °C water bath for 13 min, and then allowed to recover at 22 °C for another week before they were photographed and calculate their survival rate. Consistent with the expression of endogenous AtANN1 level, ann1–2 and ann1–3 seedlings were hypersensitive to heat-shock treatment, whereas heat sensitivity of ann1–1 seedlings was similar to wild-type plants (Fig. 3C, 3D). There are eight members of the Arabidopsis annexin gene family. Phylogenetic analysis indicates that AtANN1, AtANN2, and AtANN6 are all closely related, with AtANN2 and AtANN6 having 75% identity at the amino acid level (20Clark G.B. Morgan R.O. Fernandez M.-P. Roux S.J. Evolutionary adaptation of plant annexins has diversified their molecular structures, interactions, and functional roles.New Phytol. 2012; 196: 695-712Crossref PubMed Scopus (101) Google Scholar). There is expression and immunolocalization evidence suggesting that AtANN1 and AtANN2 have both distinct and overlapping functions in Arabidopsis (21Clark G.B. Sessions A. Eastburn D.J. Roux S.J. Differential expression of members of the annexin multigene family in Arabidopsis.Plant Physiol. 2001; 126: 1072-1084Crossref PubMed Scopus (129) Google Scholar, 22Clark G.B. Lee D. Dauwalder M. Roux S.J. Immunolocalization and histochemical evidence for the association of two different Arabidopsis" @default.
• W2157017347 created "2016-06-24" @default.
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• W2157017347 date "2015-03-01" @default.
• W2157017347 modified "2023-09-29" @default.
• W2157017347 title "Proteomic Study of Microsomal Proteins Reveals a Key Role for Arabidopsis Annexin 1 in Mediating Heat Stress-Induced Increase in Intracellular Calcium Levels" @default.
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