Abstract
Chloroplast-localized small heat-shock proteins (Cp-sHSP) protect Photosystem II and thylakoid membranes during heat and other stresses, and Cp-sHSP production levels are related to plant thermotolerance. However, to date, a paucity of Cp-sHSP sequences from C4 or CAM species, or from other extremely heat-tolerant species, has precluded an examination to determine if Cp-sHSP genes or proteins might differ among plants with photosynthetic pathways or between heat-sensitive and heat-tolerant species. To investigate this, we isolated and characterized novel Cp-sHSP genes in four plant species: two moderately heat-tolerant C4 species, Spartina alterniflora (monocot) and Amaranthus retroflexus (eudicot), and two very heat-tolerant CAM species, Agave americana (monocot) and Ferocactus wislizenii (eudicot) (respective genes: SasHSP27.12, ArsHSP26.43, AasHSP26.85 and FwsHSP27.52) by PCR-based genome walking and cDNA RACE. Analysis of these Cp-sHSPs has confirmed the presence of conserved domains common to previously examined species. As expected, the transit peptide was found to be the most variable part of these proteins. Promoter analysis of these genes revealed differences in CAM versus C3 and C4 species that were independent of a general difference between monocots and eudicots observed for the entire protein. Heat-induced gene and protein expression indicated that Cp-sHSP protein levels were correlated with thermotolerance of photosynthetic electron transport, and that in most cases protein and transcript levels were correlated. Thus, available evidence indicates little variation in the amino acid sequence of Cp-sHSP mature proteins between heat-sensitive and -tolerant species, but that variation in Cp-sHSP protein production is related to heat tolerance or photosynthetic pathway (CAM vs. C3 and C4) and is driven by promoter differences.
Key message We isolated and characterized four novel Cp-sHSP genes with promoters from wild plants, analysis has shown qualitative and quantitative interspecific variations in Cp-sHSPs of C3, C4, and CAM plant thermotolerance.
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References
Abranches R, Shultz RW, Thompson WF, Allen GC (2005) Matrix attachment regions and regulated transcription increase and stabilize transgene expression. Plant Biotechnol J 3:535–543
Amin J, Ananthan J, Voellmy R (1988) Key features of heat shock regulatory elements. Mol Cell Biol 8:3761–3769
Azzoni AR, Tada SF, Rosselli LK, Paula DP, Catani CF, Sabino AA, Barbosa JA, Guimaraes BG, Eberlin MN, Medrano FJ, Souza AP (2004) Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa. Protein Expr Purif 33:297–303
Barcala M, Garcia A, Cubas P, Almoguera C, Jordano J, Fenoll C, Escobar C (2008) Distinct heat-shock element arrangements that mediate the heat shock, but not the late-embryogenesis induction of small heat-shock proteins, correlate with promoter activation in root-knot nematode feeding cells. Plant Mol Biol 66:151–164
Barros MD, Czarnecka E, Gurley WB (1992) Mutational analysis of a plant heat shock element. Plant Mol Biol 19:665–675
Barua D, Downs CA, Heckathorn SA (2003) Variation in chloroplast small heat-shock protein function is a major determinant of variation in thermotolerance of photosynthetic electron transport among ecotypes of Chenopodium album. Funct Plant Biol 30:1071–1079
Barua D, Heckathorn SA, Coleman JS (2008) Variation in heat-shock proteins and photosynthetic thermotolerance among natural populations of Chenopodium album L. from contrasting thermal environments: implications for plant responses to global warming. J Integr Plant Biol 50:1440–1451
Basha E, Jones C, Wysocki V, Vierling E (2010) Mechanistic differences between two conserved classes of small heat shock proteins found in the plant cytosol. J Biol Chem 285:11489–11497
Carranco R, Almoguera C, Jordano J (1999) An imperfect heat shock element and different upstream sequences are required for the seed-specific expression of a small heat shock protein gene. Plant Physiol 121:723–730
Chen Q, Vierling E (1991) Analysis of conserved domains identifies a unique structural feature of a chloroplast heat shock protein. Mol Gen Genet 226:425–431
Czarnecka E, Gurley WB, Nagao RT, Mosquera LA, Key JL (1985) DNA sequence and transcript mapping of a soybean gene encoding a small heat shock protein. Proc Natl Acad Sci USA 82:3726–3730
Downs CA, Heckathorn SA (1998) The mitochondrial small heat-shock protein protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in plants. FEBS Lett 430:246–250
Downs CA, Heckathorn SA, Bryan JK, Coleman JS (1998) The methionine-rich low-molecular-weight chloroplast heat-shock protein: evolutionary conservation and accumulation in relation to thermotolerance. Am J Bot 85:175–183
Downs CA, Jones LR, Heckathorn SA (1999) Evidence for a novel set of small heat-shock proteins that associates with the mitochondria of murine PC12 cells and protects NADH:ubiquinone oxidoreductase from heat and oxidative stress. Arch Biochem Biophys 365:344–350
Eckey-Kaltenbach H, Kiefer E, Grosskopf E, Ernst D, Sandermann H Jr (1997) Differential transcript induction of parsley pathogenesis-related proteins and of a small heat shock protein by ozone and heat shock. Plant Mol Biol 33:343–350
Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984
Fernandes M, Xiao H, Lis JT (1995) Binding of heat shock factor to and transcriptional activation of heat shock genes in Drosophila. Nucleic Acids Res 23:4799–4804
Guedes RN, Zhu KY, Opit GP, Throne JE (2008) Differential heat shock tolerance and expression of heat-inducible proteins in two stored-product psocids. J Econ Entomol 101:1974–1982
Guo SJ, Zhou HY, Zhang XS, Li XG, Meng QW (2007) Overexpression of CaHSP26 in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance. J Plant Physiol 164:126–136
Hamilton EWR, Heckathorn SA (2001) Mitochondrial adaptations to NaCl. Complex I is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physiol 126:1266–1274
Heckathorn SA, Downs CA, Sharkey TD, Coleman JS (1998) The small, methionine-rich chloroplast heat-shock protein protects photosystem II electron transport during heat stress. Plant Physiol 116:439–444
Heckathorn SA, Ryan SamanthaL, Baylis JA, Wang D, Hamilton EW III, Cundiff L, Luthe DS (2002) In vivo evidence from an Agrostis stolonifera selection genotype that chloroplast small heat-shock proteins can protect photosystem II during heat stress. Funct Plant Biol 29:935–946
Heckathorn SA, Mueller JK, Laguidice S, Zhu B, Barrett T, Blair B, Dong Y (2004) Chloroplast small heat-shock proteins protect photosynthesis during heavy metal stress. Am J Bot 91:1312–1318
Helm KW, LaFayette PR, Nagao RT, Key JL, Vierling E (1993) Localization of small heat shock proteins to the higher plant endomembrane system. Mol Cell Biol 13:238–247
Jiang C, Xu J, Zhang H, Zhang X, Shi J, Li M, Ming F (2009) A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana. Plant Cell Environ 32:1046–1059
Joshi CP, Nguyen HT (1995) 5′ untranslated leader sequences of eukaryotic mRNAs encoding heat shock induced proteins. Nucleic Acids Res 23:541–549
Lee GJ, Vierling E (1998) Expression, purification, and molecular chaperone activity of plant recombinant small heat shock proteins. Methods Enzymol 290:350–365
Lee GJ, Pokala N, Vierling E (1995) Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea. J Biol Chem 270:10432–10438
Lee BH, Won SH, Lee HS, Miyao M, Chung WI, Kim IJ, Jo J (2000) Expression of the chloroplast-localized small heat shock protein by oxidative stress in rice. Gene 245:283–290
Levin JS, Thompson WF, Csinos AS, Stephenson MG, Weissinger AK (2005) Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco. Transgenic Res 14:193–206
Liu J, Shono M (1999) Characterization of mitochondria-located small heat shock protein from tomato (Lycopersicon esculentum). Plant Cell Physiol 40:1297–1304
Mamedov TG, Shono M (2008) Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein. J Plant Res 121:235–243
Mankin SL, Allen GC, Phelan T, Spiker S, Thompson WF (2003) Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3′ MAR. Transgenic Res 12:3–12
Maqbool A, Abbas W, Rao AQ, Irfan M, Zahur M, Bakhsh A, Riazuddin S, Husnain T (2010) Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum. Biotechnol Prog 26:21–25
Nagao RT, Czarnecka E, Gurley WB, Schoffl F, Key JL (1985) Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene family. Mol Cell Biol 5:3417–3428
Nakamoto H, Suzuki N, Roy SK (2000) Constitutive expression of a small heat-shock protein confers cellular thermotolerance and thermal protection to the photosynthetic apparatus in cyanobacteria. FEBS Lett 483:169–174
Neta-Sharir I, Isaacson T, Lurie S, Weiss D (2005) Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation. Plant Cell 17:1829–1838
Osteryoung KW, Vierling E (1994) Dynamics of small heat shock protein distribution within the chloroplasts of higher plants. J Biol Chem 269:28676–28682
Park SY, Shivaji R, Krans JV, Luthe DS (1996) Heat-shock response in heat-tolerant and nontolerant variants of Agrostis palustris Huds. Plant Physiol 111:515–524
Park S-Y, Chang K-C, Shivaji R, Luthe DS (1997) Recovery from heat shock in heat tolerant and nontolerant variants of creeping bentgrass. Plant Physiol 115:229–240
Pelham HR, Bienz M (1982) A synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene. EMBO J 1:1473–1477
Prandl R, Schoffl F (1996) Heat shock elements are involved in heat shock promoter activation during tobacco seed maturation. Plant Mol Biol 31:157–162
Preczewski PJ, Heckathorn SA, Downs CA, Coleman JS (2000) Photosynthetic thermotolerance is quantitatively and positively correlated with production of specific heat-shock proteins among nine genotypes of Lycopersicon (tomato). Photosynthetica 38:127–134
Raschke E, Baumann G, Schoffl F (1988) Nucleotide sequence analysis of soybean small heat shock protein genes belonging to two different multigene families. J Mol Biol 199:549–557
Sabehat A, Lurie S, Weiss D (1998) Expression of small heat-shock proteins at low temperatures. A possible role in protecting against chilling injuries. Plant Physiol 117:651–658
Sage RF, Pearcy RW, Seemann JR (1987) The nitrogen use efficiency of C(3) and C(4) plants: III. Leaf nitrogen effects on the activity of carboxylating enzymes in Chenopodium album (L.) and Amaranthus retroflexus (L.). Plant Physiol 85:355–359
Sarkar NK, Kim YK, Grover A (2009) Rice sHsp genes: genomic organization and expression profiling under stress and development. BMC Genomics 10:393
Sato Y, Yokoya S (2008) Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein, sHSP17.7. Plant Cell Rep 27:329–334
Scarpeci TE, Zanor MI, Valle EM (2008) Investigating the role of plant heat shock proteins during oxidative stress. Plant Signal Behav 3:856–857
Scharf KD, Siddique M, Vierling E (2001) The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins). Cell Stress Chaperones 6:225–237
Shakeel S, Heckrthorn S, Hamilton WE, Luthe DS (2010) Ecotypic variation in chloroplast small heat-shock proteins and related thermotolerance in Chenopodium album is associated with differences in promoters, transcription, and protein levels, rather than mature-protein sequence
Shakeel S, Haq NU, Heckathorn SA, Hamilton EW, Luthe DS (2011) Ecotypic variation in chloroplast small heat-shock proteins and related thermotolerance in Chenopodium album. Plant Physiol Biochem 49:898–908
Siddique M, Gernhard S, von Koskull-Doring P, Vierling E, Scharf KD (2008) The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties. Cell Stress Chaperones 13:183–197
Smith SD, Didden-Zopfy B, Nobel PS (1984) High-temperature responses of North American cacti. Ecology 65:643–651
Streatfield SJ, Magallanes-Lundback ME, Beifuss KK, Brooks CA, Harkey RL, Love RT, Bray J, Howard JA, Jilka JM, Hood EE (2004) Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics. Transgenic Res 13:299–312
Stulemeijer IJ, Joosten MH, Jensen ON (2009) Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for hsp90 isoforms. J Proteome Res 8:1168–1182
Sun W, Bernard C, van de Cotte B, Van Montagu M, Verbruggen N (2001) At-HSP17.6A, encoding a small heat-shock protein in Arabidopsis, can enhance osmotolerance upon overexpression. Plant J 27:407–415
Sun W, Van Montagu M, Verbruggen N (2002) Small heat shock proteins and stress tolerance in plants. Biochim Biophys Acta 1577:1–9
Sundby C, Harndahl U, Gustavsson N, Ahrman E, Murphy DJ (2005) Conserved methionines in chloroplasts. Biochim Biophys Acta 1703:191–202
Valcu CM, Lalanne C, Plomion C, Schlink K (2008) Heat induced changes in protein expression profiles of Norway spruce (Picea abies) ecotypes from different elevations. Proteomics 8:4287–4302
Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 42:579–620
Vierling E, Key JL (1985) Ribulose 1,5-bisphosphate carboxylase synthesis during heat shock. Plant Physiol 78:155–162
Volkov RA, Panchuk II, Schoffl F (2005) Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco. Plant Mol Biol 57:487–502
Wang D, Luthe DS (2003) Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance. Plant Physiol 133:319–327
Wang L, Zhao CM, Wang YJ, Liu J (2005) Overexpression of chloroplast-localized small molecular heat-shock protein enhances chilling tolerance in tomato plant. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 31:167–174
Wang D, Barua D, Joshi P, LaCroix J, Hamilton EW, Heckathorn SA (2008) Effects of elevated CO2 on the tolerance of photosynthesis to acute heat stress in 11 (C3, C4, and CAM) species. Am J Bot 95:1–13
Waters ER (1995) The molecular evolution of the small heat-shock proteins in plants. Genetics 141:785–795
Waters ER, Vierling E (1999) Chloroplast small heat shock proteins: evidence for atypical evolution of an organelle-localized protein. Proc Natl Acad Sci USA 96:14394–14399
Waters ER, Aevermann BD, Sanders-Reed Z (2008a) Comparative analysis of the small heat shock proteins in three angiosperm genomes identifies new subfamilies and reveals diverse evolutionary patterns. Cell Stress Chaperones 13:127–142
Waters ER, Nguyen SL, Eskandar R, Behan J, Sanders-Reed Z (2008b) The recent evolution of a pseudogene: diversity and divergence of a mitochondria-localized small heat shock protein in Arabidopsis thaliana. Genome 51:177–186
Xiao XF, Wolfe S, Demain AL (1991) Purification and characterization of cephalosporin 7 alpha-hydroxylase from Streptomyces clavuligerus. Biochem J 280(Pt 2):471–474
Zahur M, Maqbool A, Irfan M, Barozai MY, Qaiser U, Rashid B, Husnain T, Riazuddin S (2009) Functional analysis of cotton small heat shock protein promoter region in response to abiotic stresses in tobacco using Agrobacterium-mediated transient assay. Mol Biol Rep 36:1915–1921
Zhang J, Stewart JMD (2000) Economical and rapid method for extracting cotton genomic DNA. J Cotton Sci 4:193–201
Zhao CM, Liu J (2006a) Overexpression of small heat-shock protein of endoplasmic reticulum enhances resistance of tomato to tunicamycin. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 32:427–432
Zhao C, Shono M, Sun A, Yi S, Li M, Liu J (2007) Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato. J Plant Physiol 164:835–841
Zhao CM, Liu J (2006b) Overexpression of small heat-shock protein of endoplasmic reticulum enhances resistance of tomato to tunicamycin. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 32:427–432
Zhou Y, Chen H, Chu P, Li Y, Tan B, Ding Y, Tsang EW, Jiang L, Wu K, Huang S (2012) NnHSP17.5, a cytosolic class II small heat shock protein gene from Nelumbo nucifera, contributes to seed germination vigor and seedling thermotolerance in transgenic Arabidopsis. Plant Cell Rep 31:379–389
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This research was fully supported by a grant from the US National Science Foundation to SAH (#IOS-0114631) and DSL (#IOS-0114632).
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Shakeel, S.N., Ul Haq, N., Heckathorn, S. et al. Analysis of gene sequences indicates that quantity not quality of chloroplast small HSPs improves thermotolerance in C4 and CAM plants. Plant Cell Rep 31, 1943–1957 (2012). https://doi.org/10.1007/s00299-012-1307-z
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DOI: https://doi.org/10.1007/s00299-012-1307-z