Plant Mol Biol Rep (2011)29:723-732 D0I10.1007s11105-010-0285-y A cDNA Clone of BcHSP81-4 from the Sterility Line (Pol CMS)of Non-heading Chinese Cabbage (Brassica campestris ssp.chinensis) Tongkun Liu·Xilin Hou·Jingyi Zhang·Yuping Song" Shuning Zhang·Ying Li Published online:8 January 2011 C Springer-Verlag 2011 Abstract BcHSP81-4 gene,a member of heat shock Introduction proteins,was identified from a suppression subtractive hybridization cDNA library in non-heading Chinese cab- During their lifetime,plant species can be subjected to bage (Brassica campestris ssp.chinensis Makino).The various stressful environments to which they respond and deduced amino acid sequence of the BcHSP81-4 cDNA adapt by means of physiological,developmental,and revealed that it has high homology to other plant organelle biochemical changes.One of the most thoroughly charac- isoforms and similar homology to both cytoplasmic and terized is the induction of heat shock proteins (HSPs). prokaryotic HSP90s.To study the regulation of gene which are a group of universal and evolutionarily conserved expression,BcHSP81-4 genes in maintainer and sterility protein (Li et al.2009).Generally,HSPs are grouped into lines were monitored at different development stages and at five major families on the basis of their molecular size in different stress treatments.Real-time PCR was used for kilodaltons:HSP110,HSP90,HSP70,HSP60,and the low quantification of BcHSP8/-4 mRNA.These results indicate molecular weight HSP families (Scharf et al.2001).The that BcHSP81-4 is not responsive to heat shock at least at heat shock proteins contribute to various cellular processes 35C,while it is very responsive to salt and cold stress. including signal transduction and the folding,localization, And high expression of BcHSP81-4 in the bud of sterile accumulation,and degradation of protein molecules in both line suggests that it may play prominent roles in sterility of plant and animal species (Yu et al.2005). pol CMS in non-heading Chinese cabbage. Several members of the plant HSP gene family have been isolated.Plants contain a wide array of HSPs.HSPs Keywords CMS.Heat shock protein.Non-heading can be induced by a variety of environmental factors Chinese cabbage.Real-time PCR including heat(Piano et al.2005),low temperature (Taylor et al.2005),and other environmental stresses such as Abbreviations drought (Vasquez-Robinet et al.2008;Xu et al.2009), CMS Cytoplasmic male sterility salinity or flooding (Liu et al.2006;Lin et al.2010), EST Expressed sequence tag cadmium or arsenite (Milioni and Hatzopoulos 1997), HSP Heat shock protein ultraviolet light,oxidative stress(Song et al.2009),osmotic SSH Suppression subtractive hybridization stress,and pathogen infection (Swindell et al.2007). However,HSPs are not only present after stress stimulation. The same proteins have been shown to be essential components of cells and developmental processes under normal physiolog- T.Liu·X.Hou·J.Zhang·Y.Song·S.Zhang·Y.Li(☒) ical conditions (Rutherford and Lindquist 1998;Fu et al. State Key Laboratory of Crop Genetics and Germplasm 2010).For example,HSP82 gene shows strong heat Enhancement,Key Laboratory of Southern Vegetable Crop inducibility at heat shock temperatures,while the HSP81 Genetic Improvement,Ministry of Agriculture, Nanjing Agricultural University, gene is strongly expressed in the absence of heat shock during Nanjing 210095,People's Republic of China the pre-meiotic and meiotic prophase stages of pollen e-mail:yingli@njau.edu.cn development but is only mildly heat inducible in leaf tissue ②Springer
A cDNA Clone of BcHSP81-4 from the Sterility Line (Pol CMS) of Non-heading Chinese Cabbage (Brassica campestris ssp. chinensis) Tongkun Liu & Xilin Hou & Jingyi Zhang & Yuping Song & Shuning Zhang & Ying Li Published online: 8 January 2011 # Springer-Verlag 2011 Abstract BcHSP81-4 gene, a member of heat shock proteins, was identified from a suppression subtractive hybridization cDNA library in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). The deduced amino acid sequence of the BcHSP81-4 cDNA revealed that it has high homology to other plant organelle isoforms and similar homology to both cytoplasmic and prokaryotic HSP90s. To study the regulation of gene expression, BcHSP81-4 genes in maintainer and sterility lines were monitored at different development stages and at different stress treatments. Real-time PCR was used for quantification of BcHSP81-4 mRNA. These results indicate that BcHSP81-4 is not responsive to heat shock at least at 35°C, while it is very responsive to salt and cold stress. And high expression of BcHSP81-4 in the bud of sterile line suggests that it may play prominent roles in sterility of pol CMS in non-heading Chinese cabbage. Keywords CMS . Heat shock protein . Non-heading Chinese cabbage . Real-time PCR Abbreviations CMS Cytoplasmic male sterility EST Expressed sequence tag HSP Heat shock protein SSH Suppression subtractive hybridization Introduction During their lifetime, plant species can be subjected to various stressful environments to which they respond and adapt by means of physiological, developmental, and biochemical changes. One of the most thoroughly characterized is the induction of heat shock proteins (HSPs), which are a group of universal and evolutionarily conserved protein (Li et al. 2009). Generally, HSPs are grouped into five major families on the basis of their molecular size in kilodaltons: HSP110, HSP90, HSP70, HSP60, and the low molecular weight HSP families (Scharf et al. 2001). The heat shock proteins contribute to various cellular processes including signal transduction and the folding, localization, accumulation, and degradation of protein molecules in both plant and animal species (Yu et al. 2005). Several members of the plant HSP gene family have been isolated. Plants contain a wide array of HSPs. HSPs can be induced by a variety of environmental factors including heat (Piano et al. 2005), low temperature (Taylor et al. 2005), and other environmental stresses such as drought (Vasquez-Robinet et al. 2008; Xu et al. 2009), salinity or flooding (Liu et al. 2006; Lin et al. 2010), cadmium or arsenite (Milioni and Hatzopoulos 1997), ultraviolet light, oxidative stress (Song et al. 2009), osmotic stress, and pathogen infection (Swindell et al. 2007). However, HSPs are not only present after stress stimulation. The same proteins have been shown to be essential components of cells and developmental processes under normal physiological conditions (Rutherford and Lindquist 1998; Fu et al. 2010). For example, HSP82 gene shows strong heat inducibility at heat shock temperatures, while the HSP81 gene is strongly expressed in the absence of heat shock during the pre-meiotic and meiotic prophase stages of pollen development but is only mildly heat inducible in leaf tissue T. Liu : X. Hou : J. Zhang : Y. Song : S. Zhang : Y. Li (*) State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Southern Vegetable Crop Genetic Improvement, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China e-mail: yingli@njau.edu.cn Plant Mol Biol Rep (2011) 29:723–732 DOI 10.1007/s11105-010-0285-y
724 Plant Mol Biol Rep(2011)29:723-732 (Marrs et al.1993).Three HSP90-family genes have also been tester and the cDNA from maintainer flowers as driver,the isolated from Arabidopsis:HSP81-1 which is heat inducible flowers including all that at all the developmental stages. and HSP81-2 and HSP81-3 which are highly expressed under An expressed sequence tag (EST)of HSP90 was found in normal growth temperatures (Yabe et al.1994).Studies on the large-scale EST sequencing of cDNA library.The full expression of HSP genes revealed that some members have length of gene was cloned by homology cloning. been shown to be specifically expressed in a variety of A gene-specific forward primer BcHSPfl was designed development stages including embryogenesis(Segui-Simarro based on the HSP90 homologous sequences in Brassica rapa et al.2003;Marrs et al.1993),seed germination (Kotak et al. (EU186354)and Arabidopsis thaliana (NM_124982.2).The 2007),shoot and root meristematic apices (Cao et al.2009). reverse primer BcHSPrl was designed according to the EST flowers (Dafny-Yelin et al.2008),and pollen grain develop- sequence.The amplification by BcHSPfl and BcHSPrl was ment (Haralampidis et al.2002:Marrs et al.1993). performed as follows:an initial denaturation(94C,2 min), Flower development is an exclusively and tightly regulated followed by 35 cycles of denaturation(94C,30 s),anneal- process (Rolland-Lagan et al.2003).There are reports ing (58C,30 s)and extension (72C,1 min),and a final suggesting that the process of flower development is extension (72C,10 min)step.The sequences of primers accompanied by the accumulation of heat shock proteins mentioned above are listed in Table 1.All primers were (Dafny-Yelin et al.2005;Wang et al.2010).However,the synthesized in Genscript Biocompany (Nanjing,China). functional significance of their accumulation in flower is still All PCR reactions above were carried out in a 25-ul reaction obscure.In this paper,we reported the cloning and character- system:1 U Takara Ex Tag,1x Ex Taq buffer (plus Mg25), ization of the BcHSP81-4 gene which is specifically and 0.2 mM dNTP mixture,0.2 mM forward primer,0.2 mM highly expressed in sterile flowers (pol CMS)of non-heading reverse primer,and 1 ul cDNA template.PCR products were Chinese cabbage.And a quantitative study of BcHSP81-4 analyzed on 1%agarose gels,extracted with a QIA quick gel expression in leaves and flowers in different development extraction kit (QIAGEN),and cloned into pMD18-T vector stages and at different stress treatments has also been assessed (TaKaRa).The resultant recombinant plasmid was then both in maintainer and sterility plants using real-time PCR. transformed into Escherichia coli host DH5alpha.The positive transformers were screened by PCR and then sequenced in Genscript Biocompany (Nanjing,China). Materials and Methods DNA Sequence Data Base Analysis Plant Material,Growth Conditions,and Stress Treatment The BcHSP8/-4 cDNA sequence was analyzed by BioEdit Pol CMS and maintainer plants of non-heading Chinese (version 7.0.1)software package,and the deduced amino cabbage(Brassica campestris ssp.chinensis Makino)were acid sequence was predicted by the open reading frame grown on soil in a light/dark cycle of 16:8 h at 24C.For (ORF)finder at the National Center for Biotechnology heat(35C)and cold (4C)stress treatments,plants with five Information website (http://www.ncbi.nlm.nih.gov/gorf/ leaves were placed in a self-manufactured light incubator in a gorf.html).The deduced amino acid sequence was analyzed light/dark cycle of 16:8 h at treatment temperature for 10 h, by Simple Modular Architecture Research Tool (http:/ respectively.For salt stress treatments,plants were placed in smart.embl-heidelberg.de).The Compute pI/Mw (http:/ 0.1 M NaCl (Roshandel and Flowers 2009)in a light/dark www.expasy.ch/tools/pi tool.html)was used to calculate cycle of 16:8 h at 24C for 10 h.RNA was isolated from the theoretical isoelectric point(pl)and molecular weight leaves at different time intervals following each of the (Mw).The amino acid sequences of different species were treatments of heat,cold,and salt stress. obtained from the GenBank database (http://www.ncbi.nih Total RNA was also isolated from different tissues of B. gov),and a multiple sequence alignment was created with campestris ssp.chinensis Makino using an RNA Extraction DNAMAN (version 5.2.2).Subsequently,the homology Kit (Takara,Nanjing,China).These included maintainer tree was also generated by DNAMAN(version 5.2.2). (leaves,flowers of different sizes (3.5 mm))and sterility (leaves,flowers of different sizes Quantitative Analysis of BcHSP81-4 mRNA Expression (4.5mm)tissues. by Real-Time PCR PCR Amplification and Cloning of BcHSP81-4 Gene Polymerase chain reaction was carried out for real-time PCR using primers BcHSPf2 and BcHSPr2.In order to quantify the A forward subtractive cDNA library was constructed using mRNA levels of genes of interest,we evaluated the potential suppression subtractive hybridization method,which was of"house-keeping genes"as expression standards (data not performed using the cDNA from pol CMS flowers as the shown).Actin,GAPDH,or ribosomal protein genes are ②Springer
(Marrs et al. 1993). Three HSP90-family genes have also been isolated from Arabidopsis: HSP81-1 which is heat inducible and HSP81-2 and HSP81-3 which are highly expressed under normal growth temperatures (Yabe et al. 1994). Studies on the expression of HSP genes revealed that some members have been shown to be specifically expressed in a variety of development stages including embryogenesis (Seguí-Simarro et al. 2003; Marrs et al. 1993), seed germination (Kotak et al. 2007), shoot and root meristematic apices (Cao et al. 2009), flowers (Dafny-Yelin et al. 2008), and pollen grain development (Haralampidis et al. 2002; Marrs et al. 1993). Flower development is an exclusively and tightly regulated process (Rolland-Lagan et al. 2003). There are reports suggesting that the process of flower development is accompanied by the accumulation of heat shock proteins (Dafny-Yelin et al. 2005; Wang et al. 2010). However, the functional significance of their accumulation in flower is still obscure. In this paper, we reported the cloning and characterization of the BcHSP81-4 gene which is specifically and highly expressed in sterile flowers (pol CMS) of non-heading Chinese cabbage. And a quantitative study of BcHSP81-4 expression in leaves and flowers in different development stages and at different stress treatments has also been assessed both in maintainer and sterility plants using real-time PCR. Materials and Methods Plant Material, Growth Conditions, and Stress Treatment Pol CMS and maintainer plants of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) were grown on soil in a light/dark cycle of 16:8 h at 24°C. For heat (35°C) and cold (4°C) stress treatments, plants with five leaves were placed in a self-manufactured light incubator in a light/dark cycle of 16:8 h at treatment temperature for 10 h, respectively. For salt stress treatments, plants were placed in 0.1 M NaCl (Roshandel and Flowers 2009) in a light/dark cycle of 16:8 h at 24°C for 10 h. RNA was isolated from leaves at different time intervals following each of the treatments of heat, cold, and salt stress. Total RNA was also isolated from different tissues of B. campestris ssp. chinensis Makino using an RNA Extraction Kit (Takara, Nanjing, China). These included maintainer (leaves, flowers of different sizes (3.5 mm)) and sterility (leaves, flowers of different sizes (4.5 mm)) tissues. PCR Amplification and Cloning of BcHSP81-4 Gene A forward subtractive cDNA library was constructed using suppression subtractive hybridization method, which was performed using the cDNA from pol CMS flowers as the tester and the cDNA from maintainer flowers as driver, the flowers including all that at all the developmental stages. An expressed sequence tag (EST) of HSP90 was found in large-scale EST sequencing of cDNA library. The full length of gene was cloned by homology cloning. A gene-specific forward primer BcHSPf1 was designed based on the HSP90 homologous sequences in Brassica rapa (EU186354) and Arabidopsis thaliana (NM_124982.2). The reverse primer BcHSPr1 was designed according to the EST sequence. The amplification by BcHSPf1 and BcHSPr1 was performed as follows: an initial denaturation (94°C, 2 min), followed by 35 cycles of denaturation (94°C, 30 s), annealing (58°C, 30 s) and extension (72°C, 1 min), and a final extension (72°C, 10 min) step. The sequences of primers mentioned above are listed in Table 1. All primers were synthesized in Genscript Biocompany (Nanjing, China). All PCR reactions above were carried out in a 25-μl reaction system: 1 U Takara Ex Taq, 1× Ex Taq buffer (plus Mg2+), 0.2 mM dNTP mixture, 0.2 mM forward primer, 0.2 mM reverse primer, and 1 μl cDNA template. PCR products were analyzed on 1% agarose gels, extracted with a QIA quick gel extraction kit (QIAGEN), and cloned into pMD18-T vector (TaKaRa). The resultant recombinant plasmid was then transformed into Escherichia coli host DH5alpha. The positive transformers were screened by PCR and then sequenced in Genscript Biocompany (Nanjing, China). DNA Sequence Data Base Analysis The BcHSP81-4 cDNA sequence was analyzed by BioEdit (version 7.0.1) software package, and the deduced amino acid sequence was predicted by the open reading frame (ORF) finder at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html). The deduced amino acid sequence was analyzed by Simple Modular Architecture Research Tool (http:// smart.embl-heidelberg.de). The Compute pI/Mw (http:// www.expasy.ch/tools/pi_tool.html) was used to calculate the theoretical isoelectric point (pI) and molecular weight (Mw). The amino acid sequences of different species were obtained from the GenBank database (http://www.ncbi.nih. gov), and a multiple sequence alignment was created with DNAMAN (version 5.2.2). Subsequently, the homology tree was also generated by DNAMAN (version 5.2.2). Quantitative Analysis of BcHSP81-4 mRNA Expression by Real-Time PCR Polymerase chain reaction was carried out for real-time PCR using primers BcHSPf2 and BcHSPr2. In order to quantify the mRNA levels of genes of interest, we evaluated the potential of “house-keeping genes” as expression standards (data not shown). Actin, GAPDH, or ribosomal protein genes are 724 Plant Mol Biol Rep (2011) 29:723–732
Plant Mol Biol Rep (2011)29:723-732 725 Table 1 Nucleotide sequences of primers used in polymerase Primer Direction sequence (5'to 3) chain reaction BcHSP90fl F ATGGCCGACGCCGAGACTTT,ORF cloning BcHSP90rl R TTAGTCGACCTCCTCCATCT,ORF cloning BcHSP90f2 F GTTAGCATGATTGGGCAGTT,for RT-PCR BcHSP90r2 R TCCTTCCTCGTCCTTCTTCT,for RT-PCR GAPDHFI CCACTAACTGCCTTGCTCCAC.for RT-PCR GAPDHRI R GCTTGCCCTCAGATTCCTCCT.for RT-PCR frequently used for this purpose,although they also demon- ORF encoding 699 amino acids.The predicted Mw of strate developmentally and stress-inducible variation of BcHSP81-4 is 80.05 kDa,and theoretical p/is 4.85.Three mRNA levels (Volkov et al.2003).In this paper,we take conserved functional domains were found in BcHSP81-4:N GAPDH as an internal standard for RT-PCR.Every gene has domain with adenosine triphosphate(ATP)-binding site(1- three replicates for real-time PCR. 211),middle domain with some client proteins such as p53 Total RNA were isolated from different tissues of B. (M domain,229-517),and C domain responsible for campestris ssp.chinensis Makino after different treatments dimerization (524-699;Muller et al.2004;Brown et al. using RNA Extraction Kit (Takara).RNA quantity was 2007).Five conserved HSP90 protein family signatures measured spectrophotometrically,and its quality was were found in the deduced amino acid sequence of checked by agarose gel electrophoresis (Sambrook et al. BcHSP81-4 (Fig.1):NKEIFLRELISNSSDALDKIR (27- 1989).Only RNA without detectable degradation of 26S 47),LGTIARSGT(94-102),and IGQFGVGFYSAYLVAD rRNA was used for subsequent preparation of poly (A)- (118-133)were located at N domain,and IKLYVRRVFI mRNA.One hundred nanograms was converted into cDNA (332-341)and GIVDSEDLPLNISRET (358-373)were using anchored Oligo dT-primer and PrimeScriptTM RT located in the M domain.The conserve "GXXGXG"motif Enzyme MixIfrom PrimeScriptTM RT Reagent Kit(Takara).was found in M domain.The C-terminal-conserved The amount of poly (A)+-mRNA/cDNA double stranded MEEVD motif was also found in the BcHSP81-4 sequence products obtained after reverse transcription was used as (Fig.2d). template preparation for real-time PCR. The alignment of the HSP90s observed show that non- The real-time PCR was performed in 25 ul of reaction heading Chinese cabbage BcHSP81-4 protein has a high mixture composed of cDNA and master mix(12.5 ul SYBR degree of sequence homology with HSP90s from other Premix Ex Tag (2x),1 ul gene forward primer(10 uM),1 ul species (Fig.3).BcHSP81-4 showed a much greater gene reverse primer(10 uM),2 ul cDNA,and ddH2O up to similarity (93%and 97%,respectively)to those of B.rapa 25 ul)using an iCycler iQ system(Bio-Rad,Hercules,CA, HSP81-4 and A.thaliana HSP81-2.Three functional USA).Amplification of PCR products was monitored via domains (N.M.and C domain)were shown to be highly intercalation of SYBR-Green (Takara).The following pro- conserved(Fig.2).The alignment result showed that the N gram was applied:initial polymerase activation 95C,30 s; and M domain segment links were variable among HSP90s then 40 cycles at 95C.20 s:52C.20 s:72C.20 s:and of different species(Fig.2b).About 20 residues at the head final extension was 72C for 10 min.In order to confirm that of the conserved "MEEVD"motif were shown to be only one specific PCR product was amplified,a melt cycle, variable (Fig.2d).The homology tree was consistent with in which PCR product was denatured from 65C to 95C, the alignment result. was added to each thermal profile to produce the melt curves.To analyze expression profile of BcHSP81-4,the Tissue Development Distribution of BcHSP8/-4 mRNA comparative Cr value method (Livak and Schmittgen 2001) was employed.The baselines were set automatically by the In flowers compared to leaves,mRNA level of software taking consistency into account. BcHSP8/-4 was increased.To different extent in different stages of development,the BcHSP81-4 gene transcript was detected at low levels in maintainer line.But in sterile Results line,the expression of BcHSP81-4 gene is very high at S2 (0.6 mm<buds<1.8 mm)and S1 (buds<0.6 mm), cDNA Cloning and Sequence Analysis of BcHSP81-4 which is about 4-to 5-fold that of buds at other stage (Fig.4).The expression of BcHSP81-4 mRNA has not a The full ORF of BcHSP81-4 cDNA sequence was obtained wide range of changes between two lines except S1 and by PCR amplification.BcHSP81-4 contains a 2,100-bp S2. ②Springer
frequently used for this purpose, although they also demonstrate developmentally and stress-inducible variation of mRNA levels (Volkov et al. 2003). In this paper, we take GAPDH as an internal standard for RT-PCR. Every gene has three replicates for real-time PCR. Total RNA were isolated from different tissues of B. campestris ssp. chinensis Makino after different treatments using RNA Extraction Kit (Takara). RNA quantity was measured spectrophotometrically, and its quality was checked by agarose gel electrophoresis (Sambrook et al. 1989). Only RNA without detectable degradation of 26S rRNA was used for subsequent preparation of poly (A) + - mRNA. One hundred nanograms was converted into cDNA using anchored Oligo dT-primer and PrimeScript™ RT Enzyme MixIfrom PrimeScript™ RT Reagent Kit (Takara). The amount of poly (A) + -mRNA/cDNA double stranded products obtained after reverse transcription was used as template preparation for real-time PCR. The real-time PCR was performed in 25 μl of reaction mixture composed of cDNA and master mix (12.5 μl SYBR Premix Ex Taq (2×), 1 μl gene forward primer(10 μM), 1 μl gene reverse primer(10 μM), 2 μl cDNA, and ddH2O up to 25 μl) using an iCycler iQ system (Bio-Rad, Hercules, CA, USA). Amplification of PCR products was monitored via intercalation of SYBR-Green (Takara). The following program was applied: initial polymerase activation 95°C, 30 s; then 40 cycles at 95°C, 20 s; 52°C, 20 s; 72°C, 20 s; and final extension was 72°C for 10 min. In order to confirm that only one specific PCR product was amplified, a melt cycle, in which PCR product was denatured from 65°C to 95°C, was added to each thermal profile to produce the melt curves. To analyze expression profile of BcHSP81-4, the comparative CT value method (Livak and Schmittgen 2001) was employed. The baselines were set automatically by the software taking consistency into account. Results cDNA Cloning and Sequence Analysis of BcHSP81-4 The full ORF of BcHSP81-4 cDNA sequence was obtained by PCR amplification. BcHSP81-4 contains a 2,100-bp ORF encoding 699 amino acids. The predicted Mw of BcHSP81-4 is 80.05 kDa, and theoretical pI is 4.85. Three conserved functional domains were found in BcHSP81-4: N domain with adenosine triphosphate (ATP)-binding site (1– 211), middle domain with some client proteins such as p53 (M domain, 229–517), and C domain responsible for dimerization (524–699; Muller et al. 2004; Brown et al. 2007). Five conserved HSP90 protein family signatures were found in the deduced amino acid sequence of BcHSP81-4 (Fig. 1): NKEIFLRELISNSSDALDKIR (27– 47), LGTIARSGT (94–102), and IGQFGVGFYSAYLVAD (118–133) were located at N domain, and IKLYVRRVFI (332–341) and GIVDSEDLPLNISRET (358–373) were located in the M domain. The conserve “GXXGXG” motif was found in M domain. The C-terminal-conserved MEEVD motif was also found in the BcHSP81-4 sequence (Fig. 2d). The alignment of the HSP90s observed show that nonheading Chinese cabbage BcHSP81-4 protein has a high degree of sequence homology with HSP90s from other species (Fig. 3). BcHSP81-4 showed a much greater similarity (93% and 97%, respectively) to those of B. rapa HSP81-4 and A. thaliana HSP81-2. Three functional domains (N, M, and C domain) were shown to be highly conserved (Fig. 2). The alignment result showed that the N and M domain segment links were variable among HSP90s of different species (Fig. 2b). About 20 residues at the head of the conserved “MEEVD” motif were shown to be variable (Fig. 2d). The homology tree was consistent with the alignment result. Tissue Development Distribution of BcHSP81-4 mRNA In flowers compared to leaves, mRNA level of BcHSP81-4 was increased. To different extent in different stages of development, the BcHSP81-4 gene transcript was detected at low levels in maintainer line. But in sterile line, the expression of BcHSP81-4 gene is very high at S2 (0.6 mm<buds<1.8 mm) and S1 (buds<0.6 mm), which is about 4- to 5-fold that of buds at other stage (Fig. 4). The expression of BcHSP81-4 mRNA has not a wide range of changes between two lines except S1 and S2. Primer Direction sequence (5′ to 3′) BcHSP90f1 F ATGGCCGACGCCGAGACTTT, ORF cloning BcHSP90r1 R TTAGTCGACCTCCTCCATCT, ORF cloning BcHSP90f2 F GTTAGCATGATTGGGCAGTT, for RT-PCR BcHSP90r2 R TCCTTCCTCGTCCTTCTTCT, for RT-PCR GAPDHF1 F CCACTAACTGCCTTGCTCCAC, for RT-PCR GAPDHR1 R GCTTGCCCTCAGATTCCTCCT, for RT-PCR Table 1 Nucleotide sequences of primers used in polymerase chain reaction Plant Mol Biol Rep (2011) 29:723–732 725
726 Plant Mol Biol Rep(2011)29:723-732 atqgccgacgccgagacttttgcattccaggcggagatcaaccagttgctctcgctcatcatcaacaccttctacagcaacaaggagatc MA D A E T F A F Q A E I NQ LL S L II N T F Y S N K E I 91 ttccttcgtgagctcatcagtaactcctccgatgctttggacaaaatcagattcgagtcattgaccgataagagcaagctcgatggccag 31 LD K I R F E S L T D KS K LD G O 181 cctgagcttttcatccacatcattccagacaaggctgacaacaccttgaccattagcgatagcggtatcggtatgaccaaggctgatttg 61 P EL F I HI I PD K A D NTL T I S D S GI GMT K A D L 271 gtcaacaaccttggaaccattgcaaggtctggtactaaagagtttatggaggcgttggctgctggagctgatgttagcatgattgggcag 91 UN N L G A RS G T K L A G A D U S M IG O 361 tttggtgttggtttctactctgcttacttggttgctgacaaggttattgtgactactaaacacaatgacgacgagcagtacgtgtgggag 121 F G U G F Y S A Y L U A D K U I U TT K H N DD E 0 YU W E 451 tctcaggctggtgggtctttcactgtgaccagagacacctctggagagagtcttggtaggggtactaagatggtccttcacctcaaggag 151 0 A G G F T UT R D T S G SL G R G T K M U L 541 gaccagttggagtaccttgaggagaggaggcttaaggatttggtcaagaagcactctgagttcatcagctacccaatctccctgtggatc 181 DQ L E Y L EE RR L K D KK H S F I S YP L W I 631 gagaagaccattgagaaggagattagtgatgacgaagatgatgaagagaagaaggacgaggaaggaaaggttgaggaggtcgatgaggag 211 E K T I E K E IS DD E DD EE KK D EE G K U EE U D EE 721 aaagaaaaggaggagaagaaaaagaagaagataaaggaggtttctegegagtgggacttggtgaacaagcagaagcctatctggatgagg 241 K E K EE KKKKK I K E U S R EW D L UN K OK P 811 aagcctgaggagatcaacaaagaggagtacgctgccttctacaagagtctcagcaacgattgggaagagcatttggccgtgaagcacttc 271 K P EE I N K E E Y AA F Y K S L S N D W EE H L A U K H F 901 tcggttgagggacagctcgagttcaaggctgttctctttgtacccaagagagctccttttgatctatttgacaccaagaagaagcccaac 301 S U E G O L E F K A U L F U P K R A P F D L F D T KKK P N 991 aacatcaagctttacgtccgtcgtgtcttcatcatggacaactgtgaggacatcatcccagagtaccttggctttgtcaaaggtattgtc 331 N I K L Y U RR U F I M D N C E D II P EY L G F U K G I U 1081 gactctgaagatcttcctctcaacatctcaagagagacgttgcagcagaacaagatccttaagqtcatccqcaagaacctcqtgaagaag 361 D S E D L P L QQ N K I L KUI R K N L KK 1171 391 YN K F Y 1261 cacgaggactctcagaacagaaccaagatcgctgagctgctccgttaccactcgaccaagagcggtgatgagttgaccagcctcaaggat 421 H E D S 0 N R T K I A E LL R Y H S T K S G D E L T S LK D 1351 tacgtgacaaggatgaaggaaggtcaggaggatatcttctacatcaccggtgagagcaaaaaggctgtggagaattctccattccttgag 51 YUT R MK E I F YI T KK A U E N S P F L E 1441 aggctcaagaagaaaggctatgaagtcctttacatggttgatgccatcgatgagtatgccattggccagctcaaggaattcgagggaaag 481 R L KKK G Y E U L Y M U D A I D E Y A I G Q L K E F E G K 1531 aagctcgtctctgcaaccaaggaaggcttgaaattggaagagtcagaggacgagaagaagaaaaaggaggagctcaaggagaagtttgag 511 K L U S A T K E G L K L E E D E K KK EE L K E E 1621 ggactctgcaaagtgatcaaggacgtacttggagacaaggtegagaaggttattgtatccbaccgtgttgtggactegccttgctgtctg 541 G L C K U I K D U L G D K U E K U I U S D R UU D S P CC L 1711 gtaacaggggaatacggctggactgctaacatggagaggatcatgaaagcgcaggctttgagagacagcagcatgggaggttacatgtcc 571 U T G E Y G T A N H E R I M K AO A LR D SSM GG YM S 1801 agcaagaagacaatggagatcaatcctgagaatgcgatcatggacgagctgaggaagagagccgaagctgacaagaatgacaagtctgtg 601 S K KT M E I N P E N A I M D E L R K R A E A D K N D K S U 1891 aaggatcttgttcttctcctctttgagactgctcttctcacttcaggtttcagcttagacgagcccaacaccttcqgaagcagaatccac 631 K D L U LLL F E T A LL T S G F S L D E P N T F G S R I H 1981 aggatgttgaagctgggattgagcattgatgatgatgatgccgttgaagccgatgcagagatgccaccgcttgaggatgatgccgatgct 661 R M L K L G L S I DDDD A U E A D A E M PP L E DD A D A 2071 gaaggcagtaagatggaggaggtcgactaa 691 EG S KM EE U D Fig.1 Complete ORF of BcHSP81-4 cDNA and deduced amino acid Conserved "GxxGxG"motif is shaded with gray background. sequences.Start codon (ATG)is boxed.Asterisk indicates stop codon Conserved "MEEVD"motif is double-underlined (TAA).Five HSP90 protein family signatures are underlined Expression Profile of BcHSP81-4 mRNA Under Heat Expression of BcHSP81-4 mRNA Under Salt Stress and Cold Shock Conditions Conditions BcHSP81-4 mRNA levels showed no significant changes Considering that the expressions of some members of the during 8 h after heat treatment at 35C both in sterile and HSP90 gene family could be induced by salt stress in A. maintainer lines,although the expression level of BcHSP8/-4 thaliana (Yabe et al.1994),we watered the plants using mRNA has a significant deviation at 10 h in both lines. higher concentration (0.1 M)of NaCl.The result showed While after induced at 4C.the mRNA levels of BcHSP8/-4 that the mRNA synthesis of BcHSP81-4 was also induced gene in sterile plants was strongly and quickly increased at by salt stress.At 4 h during salt stress,the transcription 2 h,which was shown to be 8-fold higher than that of the levels of BcHSP81-4 were greatly downregulated (10-fold) control at the starting point,then it restored to the normal in both sterile and maintainer plants.However,in main- condition at 4 h and maintain the level.But in maintainer tainer plants,it slowly restored to the normal level at 10 h; plants,the expression level of BcASP8/-4 mRNA has an in sterile plants,the mRNA levels of BcHSP81-4 gene opposite distribution compare to sterile plants(Fig.5b). quickly restored to the normal level at 6 h and greatly ≌Springer
Expression Profile of BcHSP81-4 mRNA Under Heat and Cold Shock Conditions BcHSP81-4 mRNA levels showed no significant changes during 8 h after heat treatment at 35°C both in sterile and maintainer lines, although the expression level of BcHSP81-4 mRNA has a significant deviation at 10 h in both lines. While after induced at 4°C, the mRNA levels of BcHSP81-4 gene in sterile plants was strongly and quickly increased at 2 h, which was shown to be 8-fold higher than that of the control at the starting point, then it restored to the normal condition at 4 h and maintain the level. But in maintainer plants, the expression level of BcHSP81-4 mRNA has an opposite distribution compare to sterile plants (Fig. 5b). Expression of BcHSP81-4 mRNA Under Salt Stress Conditions Considering that the expressions of some members of the HSP90 gene family could be induced by salt stress in A. thaliana (Yabe et al. 1994), we watered the plants using higher concentration (0.1 M) of NaCl. The result showed that the mRNA synthesis of BcHSP81-4 was also induced by salt stress. At 4 h during salt stress, the transcription levels of BcHSP81-4 were greatly downregulated (10-fold) in both sterile and maintainer plants. However, in maintainer plants, it slowly restored to the normal level at 10 h; in sterile plants, the mRNA levels of BcHSP81-4 gene quickly restored to the normal level at 6 h and greatly Fig. 1 Complete ORF of BcHSP81-4 cDNA and deduced amino acid sequences. Start codon (ATG) is boxed. Asterisk indicates stop codon (TAA). Five HSP90 protein family signatures are underlined. Conserved “GxxGxG” motif is shaded with gray background. Conserved “MEEVD” motif is double-underlined 726 Plant Mol Biol Rep (2011) 29:723–732
Plant Mol Biol Rep (2011)29:723-732 727 Fig.2 Homology analysis of a Bchsp81-4 MADAETFAFQAE INQLLSLIINTFYSNKE IFLREL ISNS BcHSP81-4 deduced amino ac- B.rapa hspS1-4 id sequence aligned by Bioedit 7.0.a The alignment of N A.thaliana hsp81-2 MADAETFAFQAE INQLLSLIINTFYSNKE IFLREL ISNS domain of BcHSP81-4.Three A.thaliana hsp 81-3 MADAETF AFQAE INQLLSLIINTFYSNKEIFLREL ISNS HSP90 protein family signatures A.thaliana hsp81-4 MADAETF AFQAE INQLLSLIINTFYSNKE IFLREL ISNS are shaded with gray back- A.thaliana hsp90 MADAETFAFQAE INQLLSLI INTFYSNKE IFLREL ISNS ground.b The link segment H.vulgare hsp90 MATETETFAFQAE INOLLSLI INTFYSNKE IFLREL ISNS between N and M domain.c The Nbenthamiana hsp90 MAEAETF AFQAE INQLLSLIINTFYSNKE IFLRELISNS alignment of M domain of T.aestivum hsp90 HATETETF AFQAE INQLLSLI INTFYSNKE IFLRELISNS BcHSP8/-4.Two HSP90 pro- V.pseudoreticulata hsp90 MAETETF AFQAE INQLLSLIINTFYSNKE IFLREL ISNA tein family signatures are shad- Zea hsp90 MASETETF AFQAE INQLLSLIINTFYSNKE IFLREL ISNS ed with gray background.d The alignment of C terminus of BcHSP81-4.The residues shad- Bchsp81-4 SDALDKIRFESLTDKSKLDGQPELFIHIIPDKADNTLTIS ed are variable parts located at B.rapa hsp81-4 the C terminus of BcHSP81-4. A.thaliana hsp81-2 SDALDKIRFESLTDKSKLDGQPELF IHIIPDKTNNTLTII The conserved"MEEVD"motif A.thaliana hsp81-3 SDALDKIRFESLTDKSKLDGOPELFIHIIPDKTNNTLTII is boxed.The variable part is A.thaliana hsp 81-4 SDALDKIRFESLTDKSKLDGQPELF IHIIPDKTNNTLTII shaded with gray background. A.thaliana hsp90 SDALDKIRFESLTDKSKLDGQPELF IHIIPDKTNNTLTII Dots indicate at the point that all H.yulgare hsp90 SDALDKIRFESLTDKSKLDAQPELFIHIIPDKATSTLTIV residues are nonexistent.Acces- Nbenthamiana hsp90 SDALDKIRFESLTDKSKLDAQPELFIHIIPDKTNNTLTII sion numbers are shown as T.aestiyum hsp90 SDALDKIRFESLTDKSKLDAQPELFIHIIPDKATNTLTIV follows:Bchsp81-4,B.cam- pestris ssp.chinensis Makino V.pseudoreticulata hsp90 SDALDKIRFESLTDKSKLDAQPELFIHIIPDKTNNSLTII HSP81-4;B.rapa hsp81-4, Zea hsp90 SDALDKIRFESLTDKTKLDAQPELF IHIVPDKANNTLTII EU186354;A.thaliana hsp81-3, NM 124983.3;A.thaliana Bchsp81-4 DSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADVSMIG hsp81-4,NM124982.2:A. B.rapa hsp$1-4 ....MTKADLVNNLGTIARSGTKEFMEALAAGAGVSMIG thaliana hsp90,AY081302.1; A.thaliana hsp 81-2 DSGIGMTKADLVNNLGTIARSGTKEFHEALAAGADVSMIG Hordeum vulgare hsp90, AY325266.1:Nicotiana ben- A.thaliana hsp81-3 DSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADVSMIG thamiana hsp90,AY368905.1; A.thaliana hsp 81-4 DSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADVSMIG Triticum aestivum hsp90. A.thaliana hsp90 DSGIGMTKADLVNNLGTIARSGTKEFHEALAAGADVSMIG DQ665784.1:Vitis pseudoreti- H.vulgare hsp90 DSGIGMTKSDLVNNLGTIARSGTKEFMEALAAGADVSMIG culata hsp90.EU239815.1:Zea Nbenthamiana hsp90 DSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADVSMIG hsp90,FJ805746.1 T.aestivum hsp90 DSGIGMTKSDLVNNLGTIARSGTKEFMEALAAGADVSMIG V.pseudoreticulata hsp90 DSGIGMTKADLVNNLGTIARSGTKEFHEALAAGADVSMIG Zea hsp90 DSGIGMTKSDLVNNLGTIARSGTKEFMEALAAGADVSMIG Bchsp81-4 QF GVGFYSAYLVADKVIVTTKHNDDEQYVUESQAGGSFTV B.rapa hsp81-4 QF GVGFYSAYLVADKVIVTTKHNDDEQYVUESQAGGSFTV A.thaliana hsp81-2 QF GVGFYSAYLVADKVVVTTKHNDDEQYVUESQAGGSFTV A.thaliana hsp81-3 QF GVGFYSAYLVADKVVVTTKHNDDEQYVUESQAGGSFTV A.thaliana hsp81-4 QF GVGFYSAYLVADKVVVTTKHNDDEQYVUESQAGGSFTV A.thaliana hsp90 OF GVGFYSAYLVADKVVVTTKHNDDEQYVUESQAGGSFTV H.yulgare hsp90 QF GVGFYSAYLVAERVVVTTKHNDDEQYVUESQAGGSFTV N.benthamiana hsp90 QFGVGFYSAYLVAERVVVTTKHNDDEQYVUESQAGGSFTV T.aastivum hsp90 QF GVGFYSAYLVAERVVVTTKHNDDEQYVUESQAGGSFTV V.pseudoreticulata hsp90 QF GVGFYSAYLVAEKVIVTAKHNDDEQYVUESQAGGSFTV Zea hsp90 QF GVGFYSAYLVAERVVVTTKHNDDEQYVUESQAGGSFTV Bchsp814 TRDTSGESLGRGTKMVLHLKEDQLEYLEERRLKDLVKKH B.rapa hsp81-4 TRDTSGESLGRGTKMVLHLKEDQLEYLEERRLNDLVKKH A.thaliana hsp81-2 TRDTSGETLGRGTKMVLYLKEDQLEYLEERRLKDLVKKH A.thaliana hsp81-3 TRDTSGEALGRGTKMVLYLKEDQMEYIEERRLKDLVKKH A.thaliana hs里814 TRDTSGEALGRGTKMILYLKEDOMEYIEERRLKDLVKKH A.thaliana hsp90 TRDTSGEALGRGTKMVLYLKEDOMEYIEERRLKDLVKKH H.vulgare hsp90 TRDTSGEQLGRGTKMVLYLKDDQMEYLEERRIKDLVKKH benthamiana hs里90 TRDTSGENLGRGTKITLFLKEDQLEYLEERRLKDLVKKH T.aestivum hsp90 TRDTSGEQLGRGTKMVLYLKDDQMEYLEERRIKDLVKKH V.pseudoreticulata hsp90 TRDTSGESLGRGTKITLYLKEDQLEYLEERRVKDLIKKH Zea hsp90 ARDTSGEQLGRGTKMTLYLKDDQLEYLEERRLKDLIKKH ②Springer
Fig. 2 Homology analysis of BcHSP81-4 deduced amino acid sequence aligned by Bioedit 7.0. a The alignment of N domain of BcHSP81-4. Three HSP90 protein family signatures are shaded with gray background. b The link segment between N and M domain. c The alignment of M domain of BcHSP81-4. Two HSP90 protein family signatures are shaded with gray background. d The alignment of C terminus of BcHSP81-4. The residues shaded are variable parts located at the C terminus of BcHSP81-4 . The conserved “MEEVD ” motif is boxed. The variable part is shaded with gray background . Dots indicate at the point that all residues are nonexistent. Accession numbers are shown as follows: Bchsp81-4 , B. campestris ssp. chinensis Makino HSP81-4 ; B. rapa hsp81-4 , EU186354; A. thaliana hsp81-3 , NM_124983.3; A. thaliana hsp81-4, NM_124982.2; A. thaliana hsp90, AY081302.1; Hordeum vulgare hsp90 , AY325266.1; Nicotiana benthamiana hsp90, AY368905.1; Triticum aestivum hsp90 , DQ665784.1; Vitis pseudoreticulata hsp90, EU239815.1; Zea hsp90, FJ805746.1 Plant Mol Biol Rep (2011) 29:723 –732 727
728 Plant Mol Biol Rep(2011)29:723-732 Fig.2 (continued) b Bcksp814 SEFISYPISLUIEKTIEKEISDDEDDEEKKD.EEGKVEEV B.rapa hsp81-4 SEFISYPISLWIEKTIEKRLVMTKMMREEAR.KER..LRV A.thaliana hsp 81-2 SEFISYPISLUIEKTIEKEISDDEEEEEKKD.EEGKVEEV A.thaliana hsp81-3 SEFISYPISLWIEKTIEKEISDDEEEEEKKD.EEGKVEEV A.thaliana hsp81-4 SEFISYPISLWIEKTIEKEISDDEEEEEKKD.EEGKVEEI A.thaliana hsp90 SEFISYPISLUIEKTIEKEISDDEEEEEKKD.EEGKVEEV H.vulgare hsp90 SEFISYPISLUTEKTTEKEISDDEDEEEKKDTEEGKVEDV benthamiana hs罗90 SEFISYPISLUVEKTIEKEISDDEDEEEKKD.EEGKVGEV T.aestivum hsp90 SEFISYPISLUTEKTTEKEISDDEDEEEKKDTEEGKVEDV V.pseudoreticulata hsp90 SEFISYPISLWIEKTTEKEISDDEDEEDKKD.EEGKVEEV Zea hsp90 SEFISYPISLUIEKTTEKEISDDEDEEDKKD.EEGKVEDV Bchsp81-4 DEEKEKEEKKKKKI B.rapa hsp81-4 DEEKEKEEKKKKKI A.thaliana hsp81-2 DEEKEKEEKKKKKI A.thaliana hsp81-3 DEEKEKEEKKKKKI A.thaliana hsp$1-4 DEEKEKEEKKKKKI A.thaliana hsp90 DEEKEKEEKKKKKI H.vulgare hsp90 DEEKEEKEKKKKKI Nbenthamiana hsp90 DEEKEKEEKKKKKI T.aestivum hsp90 DEEKEEKEKKKKKI V.pseudoreticulata hsp90 DEEKEKEEKKKKKI Zea hsp90 DDEKEEKEKKKKKI C Bcksp81-4 NNIKLYVRRVFIMDNCEDIIPEYLGFVKGIVDSEDLPLNI B.ra叹a理814 NNIKLYVRRVF IMDNCEDIIPEYLGFVKGIVDSEDLPLNI A.thaliana hsp81-2 NNIKLYVRRVF IMDNCEDIIPEYLGFVKGIVDSEDLPLNI A.thaliana hsp81-3 NNIKLYVRRVF IMDNCEDIIPEYLGFVKGIVDSEDLPLNI A.thaliana hsp81-4 NNIKLYVRRVF IMDNCEDIIPDYLGFVKGIVDSEDLPLNI A.thaliana hsp90 NNIKLYVRRVF IMDNCEDIIPEYLGFVKGIVDSEDLPLNI H.vulgare hsp90 NNIKLYVRRVF IMDNCDEL IPEYLSFVKGIVDSEDLPLNI Nbenthamiana hsp90 NNIKLYVRRVFIMDNCEEL IPEYLSFVKGIVDSEDLPLNI T.aestivum里90 NNIKLYVRRVF IMDNCEEL IPEYLSFVKGIVDSEDLPLNI V.pseudoreticulata hsp90 NNIKLYVRRVF IMDNCEEL IPEYLGFVKGIVDSEDLPLNI Zea hsp90 NNIKLYVRRVF IMDNCEELIPEWLSFVKGIVDSEDLPLNI Bcksp81-4 SRETLQQ B.rapa hsp81-4 SRETLQQ A.thaliana hsp81-2 SRETLQQ A.thaliana hsp81-3 SRETLQQ A.thaliana hsp81-4 SRETLQQ A.thaliana hsp90 SRETLQQ H.yulgare hsp90 SRETLQQ Nbenthamiana hsp90 SREMLQQ T.aestivum hs罗90 SRETLQQ V.pseudoreticulata hsp90 SREMLQQ Zea hsp90 SRETLQQ d Bcksp81-4 LGLSIDD.DDAVEADAEMPPLED.DADAEGSKMEEVD B.rapa hsp81-4 LGLSIDD.DDAVEADAEMPPLED.DADAEGSKMEEVD A.thaliana hs里81-2 LGLSIDD.DDAVEADAEMPPLED.DADAEGSKMEEVD A.thaliana hsp81-3 LGLSIDD.DDVVEADADMPPLED.DADAEGSKMEEVD A.thaliana hsp81-4 LGLSIEE.DDAVEADAEMPPLED.DADAEGSKMEEVD A.thaliana hsp90 LGLSIDD.DDVVEADADMPPLED.DADAEGSKMEEVD H.yulgare hsp90 LGLSIDEDDEAPENDTDMPPLED...DAGESKMEEVD Nbenthamiana hsp90 LGLSID..EDCGDAEADMPPLEDPEADAEGSKMEEVD T.aestivum hsp90 LGLSIDEDDEAPENDTDMPPLED...DAGESKMEEVD V.pseudoreticulata hsp90 LGLNID..EEAGDVDVDMPPLEEADAEAEGSKMEEVD Zea hsp90 LGLSIDEDEEAPEADTDMPPLED...DAGESKMEEVD 么Springer
Fig. 2 (continued) 728 Plant Mol Biol Rep (2011) 29:723 –732
Plant Mol Biol Rep (2011)29:723-732 729 100% 959% 909% was cloned from non-heading Chinese cabbage (B.cam- Beksp31-4 pestris ssp.chinensis Makino).Five conserved HSP90 family signatures were identified in the deduced BcHSP81- Brassica repa hsp81-4 4 amino acid sequence.Conserved domains of BcHSP81-4 Arabidopsis thalians hsp81-2 guarantee the conserved functions of HSP90 such as client Arabidopsis thaliana hsp81-3 protein binding (Brown et al.2007).In the C domain of BcHSP81-4,about 20 residues were highly variable among Arabidopsis thaliana hsp90 different species.The conserved "MEEVD"motif located Arabidopsis thaliana hsp81-4 at the C terminus of BcHSP81-4 indicated that BcHSP81-4 Wicotiana benthaniana hsp90 activities can probably be mediated by proteins containing tetraticopeptide repeat domains as in other species (Brown Vitis pseudoreticulata hsp90 et al.2007).In the M domain,the conserved "GXXGXG" Hordeun vulgare hsp90 motif was found,which had been predicted and which Triticum sestivum hsp90 wraps around ATP in the tertiary structure (Pearl and Prodromou 2006).The phylogeny tree revealed that Zea hsp90 BcHSP81-4 belonged to the Brassica genus and was Fig.3 The homology tree shows the relationship of BcHSP8/-4 with located in the Cruciferae family.Both the alignment result other known HSP90s.Alignment of amino acid sequences are produced by DNAMAN5.2.2,and the neighbor-joining phylogeny and the phylogeny tree analysis showed that BcHSP81-4 tree is also constructed by DNAMAN5.2.2.Bchsp81-4 is shaded with was more homologous with HSP90s of previously identi- gray background.Abbreviations and accession numbers are the same fied plants.And the protein with the right molecular weight as shown in Fig.2 could be expressed in E.coli(data not shown). Hsp is usually induced by abiotic stress,such as the upregulated (3-fold)at 10 h compared that of the control at expressions of some members of the Hsp90 gene family in the starting point(Fig.5a). A.thaliana which are induced by salt stress (Yabe et al. 1994).To see the response of BcHSP81-4 to different abiotic stress,both of lines were used.BcHSP81-4 showed Discussion responsive to salt stress and cold stress,respectively,but no responsive to heat shock at 35C.Most of HSPs are HSP90,co-chaperoned with other proteins such as the responsive to heat shock at 35C (Takahashi et al.1992: HSP70 family proteins (Pratt and Toft 2003),is a highly Yuan et al.1997)or even at lower temperature (Theodoraki conserved and abundant cytoplasmic protein involved in and Mintzas 2006).Some other reports showed that the protein folding,cytoprotection,proteosomic degradation, HSP was induced at 45C in Arabidopsis (Scarpeci et al. and a number of cellular regulatory pathways(Zhang and 2008).Maybe it was not enough for BcHSP81-4 to be Burrows 2004;Brown et al.2007).In this study,the full- induced at the temperature of 35C,and there would be a ORF cDNA sequence of cytoplasmic HSP90(BcHSP8/-4) different result at higher temperature.However,we still can say that BcHSP81-4 is no responsive to heat shock at least Ssterile line maintainer line at 35C that is different with most of HSP which showed 3.01 responsive to heat shock at 35C. 2.5 Hsp90 has a strategic role in the developmental 2.0 dynamics that govern early morphogenesis(Samakovli et 1.5 al.2007).SHD,encoding an ER-resident HSP90-like T 1.0 protein,has a function in promoting elongation of pollen 0.5 tube (Ishiguro et al.2002).In non-heading Chinese 0.0 cabbage,the development of the pol CMS anthers is FL SL F1 S1 F2 S2 F3 S3 F44 Flowers at different stages of development inhibited at the stage of archesporial cell.There is no differentiation of archesporial cell and no anther sac Fig.4 Alterations of mRNA levels for BcHSP8/-4 genes during different development stages in non-heading Chinese cabbage (B. formed.In this stage,the size of flower is about 0.5 mm campestris ssp.chinensis).FI,F2,F3,F4 maintainer flower in in non-heading Chinese cabbage (Yang and Cao 1997).In different sizes (3.5 mm);FL maintainer leaves;S1,S2, our study,the expression of BcHSP81-4 gene in sterile line S3,S4 sterility flower in different sizes(4.5 mm);SL is very high at S2(0.6 mm<buds<1.8 mm)and S1 (buds< sterility leaves.Poly (A)'-mRNA was isolated from flowers at different stages of development,converted to cDNA,and subjected to 0.6 mm),which is about 4-to 5-fold that of buds at other comparative real-time PCR quantification.Each data point represents stage (Fig.4),while in maintainer line,the expression of mcan±SE(n=3) BcHSP81-4 gene keeps lower level during the flower 鱼Springer
upregulated (3-fold) at 10 h compared that of the control at the starting point (Fig. 5a). Discussion HSP90, co-chaperoned with other proteins such as the HSP70 family proteins (Pratt and Toft 2003), is a highly conserved and abundant cytoplasmic protein involved in protein folding, cytoprotection, proteosomic degradation, and a number of cellular regulatory pathways (Zhang and Burrows 2004; Brown et al. 2007). In this study, the fullORF cDNA sequence of cytoplasmic HSP90 (BcHSP81-4) was cloned from non-heading Chinese cabbage (B. campestris ssp. chinensis Makino). Five conserved HSP90 family signatures were identified in the deduced BcHSP81- 4 amino acid sequence. Conserved domains of BcHSP81-4 guarantee the conserved functions of HSP90 such as client protein binding (Brown et al. 2007). In the C domain of BcHSP81-4, about 20 residues were highly variable among different species. The conserved “MEEVD” motif located at the C terminus of BcHSP81-4 indicated that BcHSP81-4 activities can probably be mediated by proteins containing tetraticopeptide repeat domains as in other species (Brown et al. 2007). In the M domain, the conserved “GXXGXG” motif was found, which had been predicted and which wraps around ATP in the tertiary structure (Pearl and Prodromou 2006). The phylogeny tree revealed that BcHSP81-4 belonged to the Brassica genus and was located in the Cruciferae family. Both the alignment result and the phylogeny tree analysis showed that BcHSP81-4 was more homologous with HSP90s of previously identified plants. And the protein with the right molecular weight could be expressed in E. coli (data not shown). Hsp is usually induced by abiotic stress, such as the expressions of some members of the Hsp90 gene family in A. thaliana which are induced by salt stress (Yabe et al. 1994). To see the response of BcHSP81-4 to different abiotic stress, both of lines were used. BcHSP81-4 showed responsive to salt stress and cold stress, respectively, but no responsive to heat shock at 35°C. Most of HSPs are responsive to heat shock at 35°C (Takahashi et al. 1992; Yuan et al. 1997) or even at lower temperature (Theodoraki and Mintzas 2006). Some other reports showed that the HSP was induced at 45°C in Arabidopsis (Scarpeci et al. 2008). Maybe it was not enough for BcHSP81-4 to be induced at the temperature of 35°C, and there would be a different result at higher temperature. However, we still can say that BcHSP81-4 is no responsive to heat shock at least at 35°C that is different with most of HSP which showed responsive to heat shock at 35°C. Hsp90 has a strategic role in the developmental dynamics that govern early morphogenesis (Samakovli et al. 2007). SHD, encoding an ER-resident HSP90-like protein, has a function in promoting elongation of pollen tube (Ishiguro et al. 2002). In non-heading Chinese cabbage, the development of the pol CMS anthers is inhibited at the stage of archesporial cell. There is no differentiation of archesporial cell and no anther sac formed. In this stage, the size of flower is about 0.5 mm in non-heading Chinese cabbage (Yang and Cao 1997). In our study, the expression of BcHSP81-4 gene in sterile line is very high at S2 (0.6 mm3.5 mm); FL maintainer leaves; S1, S2, S3, S4 sterility flower in different sizes (4.5 mm); SL sterility leaves. Poly (A) + -mRNA was isolated from flowers at different stages of development, converted to cDNA, and subjected to comparative real-time PCR quantification. Each data point represents mean ± SE (n=3) Fig. 3 The homology tree shows the relationship of BcHSP81-4 with other known HSP90s. Alignment of amino acid sequences are produced by DNAMAN5.2.2, and the neighbor-joining phylogeny tree is also constructed by DNAMAN5.2.2. Bchsp81-4 is shaded with gray background. Abbreviations and accession numbers are the same as shown in Fig. 2 Plant Mol Biol Rep (2011) 29:723–732 729
730 Plant Mol Biol Rep(2011)29:723-732 Nsterile line Omaintainer line a 1.2 3 Salt stress b Cold stress 2.5 0.8 0.6 1.5 1 0.2 0.5 0 0 0.5 0.8 0.4 0.6 03 0.2 04 0.1 0.2 10 0 10 Time (Hour) Time (Hour) 2.5 Heat stress 1.5 1 0.5 囹图 1.5 0 2 6 10 Time (Hour) Fig.5 Quantitative analysis of BcHSP8/-4 mRNA in different tissues maintainer and sterility lines after heat shock treatment analyzed by of maintainer and sterility lines by real-time RT-PCR.a BcHSP81-4 real-time RT-PCR.The x-axis 0,before stress treatments 2-10-2,4, relative expression in maintainer and sterility lines after salt stress 6,8,and 10 h during stress treatments (salt stress at 0.1 M NaCl;heat treatment analyzed by real-time RT-PCR.b BcHSP8/-4 relative shock at 35C;cold stress at 4C).The y-axis:relative expression of expression in maintainer and sterility lines after cold stress treatment BcHSP81-4 mRNA in different stress treatments.Each data point analyzed by real-time RT-PCR.c BcHSP81-4 relative expression in represents mean SE (n=3) development.It implied the important role of BcHSP81-4 in is almost unchanged,low level in normal flower develop- the male sterile of pol CMS in non-heading Chinese ment (Fig.4).It is highly upregulated,however,in the stage cabbage. of pre-bud formation in pol CMS (Fig.4).That also implies HSP is the center of many cellular processes including some relationship between the HSP expression and the growth,cell cycling,apoptosis,development,and evolution male sterility. (Jackson et al.2004).Most research about fertility and HSP Actually,different HSPs is responsive to different stress. showed that Hsp is essential for the normal development of The central component of the heat shock response is flower,such as bobl (small heat shock protein)mutant in oxidative stress (Haralampidis et al.2002).The mtDNA Arabidopsis exhibit reduced fertility (Perez et al.2008),and of pol cytoplasm was distinguished from the mtDNAs by many Hsps are expressed in microspores or mature pollen restriction analysis (Erickson et al.1986).Analysis of (Magnard et al.1996;Haralampidis et al.2002).HSP cybrid lines has indicated that the determinants for pol proteins play a role in protecting cell proteins at various CMS reside in the mitochondrial genome (Kemble and developmental stages (Mery et al.2008).But some also Barsby 1988).The imbalance of mitochondrial energy showed that the expression of HSP is higher in sterile line metabolism is the cause of male sterility.That cause the than that of fertile line (Sheoran et al.2009).and the oxidative stress,which was proved by the higher activities expression of Cihsp70 even is required for the switch from of POD,SOD,and CAT in CMS line than those in its self-fertility to self-sterility in Ciona intestinalis (Marino et maintainer line (Deng et al.2007).Maybe it is that al.1998).Here,in our paper,the expression of BcHSP81-4 BcHSP81-4 is responsive to the oxidative stress in ≌Springer
development. It implied the important role of BcHSP81-4 in the male sterile of pol CMS in non-heading Chinese cabbage. HSP is the center of many cellular processes including growth, cell cycling, apoptosis, development, and evolution (Jackson et al. 2004). Most research about fertility and HSP showed that Hsp is essential for the normal development of flower, such as bob1 (small heat shock protein) mutant in Arabidopsis exhibit reduced fertility (Perez et al. 2008), and many Hsps are expressed in microspores or mature pollen (Magnard et al. 1996; Haralampidis et al. 2002). HSP proteins play a role in protecting cell proteins at various developmental stages (Mery et al. 2008). But some also showed that the expression of HSP is higher in sterile line than that of fertile line (Sheoran et al. 2009), and the expression of Cihsp70 even is required for the switch from self-fertility to self-sterility in Ciona intestinalis (Marino et al. 1998). Here, in our paper, the expression of BcHSP81-4 is almost unchanged, low level in normal flower development (Fig. 4). It is highly upregulated, however, in the stage of pre-bud formation in pol CMS (Fig. 4). That also implies some relationship between the HSP expression and the male sterility. Actually, different HSPs is responsive to different stress. The central component of the heat shock response is oxidative stress (Haralampidis et al. 2002). The mtDNA of pol cytoplasm was distinguished from the mtDNAs by restriction analysis (Erickson et al. 1986). Analysis of cybrid lines has indicated that the determinants for pol CMS reside in the mitochondrial genome (Kemble and Barsby 1988). The imbalance of mitochondrial energy metabolism is the cause of male sterility. That cause the oxidative stress, which was proved by the higher activities of POD, SOD, and CAT in CMS line than those in its maintainer line (Deng et al. 2007). Maybe it is that BcHSP81-4 is responsive to the oxidative stress in Fig. 5 Quantitative analysis of BcHSP81-4 mRNA in different tissues of maintainer and sterility lines by real-time RT-PCR. a BcHSP81-4 relative expression in maintainer and sterility lines after salt stress treatment analyzed by real-time RT-PCR. b BcHSP81-4 relative expression in maintainer and sterility lines after cold stress treatment analyzed by real-time RT-PCR. c BcHSP81-4 relative expression in maintainer and sterility lines after heat shock treatment analyzed by real-time RT-PCR. The x-axis 0, before stress treatments 2–10—2, 4, 6, 8, and 10 h during stress treatments (salt stress at 0.1 M NaCl; heat shock at 35°C; cold stress at 4°C). The y-axis: relative expression of BcHSP81-4 mRNA in different stress treatments. Each data point represents mean ± SE (n=3) 730 Plant Mol Biol Rep (2011) 29:723–732
Plant Mol Biol Rep(2011)29:723-732 731 mitochondria that will result in the cell progressed death Magnard JL,Vergne P,Dumas C (1996)Complexity and genetic during flower development and then the sterility. variability of heat-shock protein expression in isolated maize microspores.Plant Physiol 111:1085-1096 Marino R,Pinto MR,Cotelli F,Lamia CL,De Santis R(1998)The hsp70 Acknowledgments This work was supported by 973 Program protein is involved in the acquisition of gamete self-sterility in the (2009CB119001-04)and Program for New Century Excellent Talents ascidian Ciona intestinalis.Development 125:899-907 in University (NCET-07-0439). Marrs KA,Casey ES,Capitant SA.Bouchard RA,Dietrich PS. 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