Abstract：Profilin(PRF) is a low molecular weight actin binding protein which is linked to the signal transduction cascade via the phosphoinositide pathways in eukaryotes, regulating both actin polymerization and depolymerization. To date, PRF has been identified in many higher plants, such as Arabidopsis thaliana, Zea mays, Akebia trifoliata, Brassica campestris. In this study, a cDNA clone(designated TaPRF7 (GenBank No. KY940299) in accordance with the accession sequence of wheat (Triticum aestivum) PRF in GenBank) encoding PRF gene was isolated from stamen of wheat photoperiod-thermo sensitive genic male sterile(PTGMS) line BS366 using RT-PCR technique. The cDNA sequence contains a 396 bp ORF, coding for 131 amino acids, with an estimated molecular mass of 14.2 kD and a pI of 4.81. Homology comparison revealed that the protein contains conservative PROF domain and actin, PIP2 and poly-L-proline (PLP) binding sites. The amino acid sequence among TaPRF7 and other different species (T. aestivum, Z. mays, Oryza sativa, Glycine max, Hordeum vulgare, Sorghum bicolor, A. thaliana, Nicotiana tabacum, Solarmum tuberosum) compared by Blast, revealing that there was high homology (70%~97%) with profiling protein of other plants and the similarity to TaPRF1(ACE82291.1) protein was the highest with 97%. Phylogenetic analysis showed that TaPRF7 has close relationship with ZmPRF2, ZmPRF4, ZmPRF5, HvPRF1, SbPRF and OsPRF. Subcellular localization analysis indicated that TaPRF7 was targeted to the nucleus and cytoplasm by detecting the fluorescent signals of eukaryotic expression vector in Arabidopsis protoplasts. Expressions of TaPRF7 had been observed in various tissues (root, stem, leaf, stamen, pistal and glume) of wheat, but was highest in stamen, followed by root, suggesting that TaPRF7 was reproductive expression and mainly expressed in reproductive organs. To investigate the involvement of TaPRF7 in signal transduction, TaPRF7 gene expression of wheat seedling in the abiotic stresses was studied. The results showed that expression of TaPRF7 was inhibited significantly by exogenous methyl jasmonate (MeJA) and salicylic acid (SA) treatments, while up-regulated under cold (10 ℃), abscisic acid (ABA), drought (PEG 6 000), gibberellin (GA), NaCl, indoleaceticacid (IAA) treatments. TaPRF7 expression speaked at different time in different abiotic treatments. The results showed that peaks appeared about 8 h after cold and NaCl treatments, 4 h after PEG and GA treatments and 2 h after IAA and ABA treatments, respectively, suggesting that TaPRF7 responses to many abiotic stresses. Further, the TaPRF7 expressions were decreased after they peaked. It was likely to be due to negative regulation which caused by accumulation of the gene. Microfilament is one component of cytoskeleton, and it is mainly composed of actin. Recent studies showed that movement of microfilament was involved in plant fertility. PRF as a microfilament-associated protein, maybe play important roles in fertility regulation. To investigate the role of TaPRF7 in fertility regulation, analysis of TaPRF7 expression profile between PTGMS line BS366 and coventional wheat line Jing411 during fertility transition was performed. It was found that TaPRF7 showed low expression and no significant change during fertility transitionin Jing411. However, the expression of TaPRF7 increased with the anther development in both sterile environment and fertile environment with the development of anther. Besides, the expressions of TaPRF7 were higher during fertility transition in sterile environment comparing with expression infertile environment. In summary, it was speculated that TaPRF7 may be involved in the signaling transduction for anther dehiscence and cold-induced male sterility associated with wheat male sterility. This study laid a basis for investigation of TaPRF7 in molecular mechanisms of wheat PTGMS line.
熊大胜, 周精华, 喻娇, 等. 2011. 三叶木通花粉前纤维蛋白基因的克隆与表达[J]. 西北植物学报, 31(8):1505-1511. (Xiong D S, Zhou J H, Yu J, et al. 2011. Cloning and expression analysis of a pollen profilin gene from Akebia trifoliata[J]. Acta Botanica Boreali-Occidentalia Sinica, 31(8):1505-1511.) 叶秋, 李旭峰, 徐莺, 等. 2001. 油菜profilin基因的克隆和表达分析[J]. 植物学报, 43(7):727-730. (Ye Q, Li X F, Xu Y, et al. 2001. Cloning and expression of a profilin gene from Rapeseed[J]. Journal of Integrative Plant Biology. 43(7):727-730.) Braun M, Balu?ka F, Witsch M V, et al. 1999. Redistribution of actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs[J]. Planta, 209(4):435-443. Braun M, Hauslage J, Czogalla A, et al. 2004. Tip-localized actin polymerization and remodeling, reflected by the localization of ADF, profilin and villin, are fundamental for gravity-sensing and polar growth in characean rhizoids[J]. Planta, 219(3):379-388. Carlsson L, Nystrom L E, Sundkvist I, et al. 1977. Actin polmerizability is influenced by profilin, a low molecular weight protein in non-muscel cells[J]. Journal of Molecular Biology, 115(3):465-483. Clarke, S R, Staiger C J, Gibbon B C, et al. 1998. A potential signaling role for profilin in pollen of Papaver rhoeas[J]. P-lant Cell, 10(6):967-979. Deeks M J, Hussey P J, Davies B. 2002. Formins: intermediates in signal-transduction cascades that affect cytoskeletal reor-ganization[J]. Trends Plant Science, 7(11):492-498. Goode B L, Eck M J. 2007. Mechanism and function of formins in the control of actin assembly[J]. Biochemistry, 76(76):593-627. Goldschmidt-Clermont P J, Kim J W, Machesky L M, et al. 1991. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation[J]. Science, 251(4998):1231-1233. Imanishi S, Nakakita M, Yamashita K, et al. 2000. Aspirin and salicylic acid do not inhibit methyl jasmonate-inducible expression of a gene for ornithine decarboxylase in tobacco BY-2 cells[J]. Agricultural and Biological Chemistry, 64(1):125-133. Kovar D R, Dr?bak B K, Staiger C J. 2000. Maize profilin isoforms are functionally distintct[J].Plant Cell, 12(4):583-598. Mandaokar A, Thines B, Shin B, et al. 2006. Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling[J]. The Plant Journal, 46(6):984-1008. Mahoney N M, Janmey P A, Almo S C. 1997. Structure of the profilin-poly-L-proline complex involved in morphogenesis and cytoskeletal regulation[J]. Nature Structural Biology, 4(11):953-960. Mckenna S T, Vidali L, Hepler P K. 2004. Profilin inhibits pollen tube growth through actin-binding, but not poly-L-proline-binding[J]. Planta, 218(6):906-915. Muruáis G, Lalioti V, Sandoval I V. 2009. The Cdk5 inhibitor roscovitine strongly inhibits glucose uptake in 3T3-L1 adipocytes without altering GLUT4 translocation from internal pools to the cell surface[J]. Journal of Cellular Physiology, 220(1):238-244. Mur L A, Kenton P, Atzorn R, et al. 2006. The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death[J]. Plant Physiology, 140(1):249-262. Ramachandran S, Christensen H E, Ismaru Y, et al. 2000. Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis[J] Plant Physiologists, 124(4):1637-1647. Romero S, Didry D, Larquet E, et al. 2007. How ATP hydrolysis controls filament assembly from profilin-actin: implication for formin processivity[J]. Journal of Biological Chemistry, 282(11):8435-8445. Spoel S H, Johnson J S, Dong X. 2007. Regulation of tradeoffs between plant defenses against pathogens with different lifestyles[J]. Proceedings of the National Academy of Sciences of the United States of America, 104(47):18842-18847. Staiger C J, Goodbody K C, Hussey P J, et al. 1993. The profilin multigene family of maize: differential expression of th-ree isoforms[J]. Plant Journal for Cell & Molecular Biology, 4(4):631-641. Staiger C J, Gibbon B C, Kovar D R, et al. 1997. Profilin and actin-depolymerizing factor: modulators of actin organizatio-n in plants[J]. Trends in Plant Science, 2(97):275–281. Staiger C J, Blanchoin L. 2006. Actin dynamics: old friends with new stories.[J]. Current Opinion in Plant Biology, 9(6):554-562. Taká? T, Pechan T, Richter H, et al. 2011. Proteomics on brefeldin a-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton[J]. Journal of Proteome Rese-arch, 10(2):488-501. Tang Z H, Zhang L P, Yang D, et al. 2011. Cold stress contributes to aberrant cytokinesis during male meiosis I in a whe-at thermosensitive genic male sterile line[J]. Plant Cell and Environment, 34(3):389-405. Valenta R, Duchêne M, Breitenbach M, et al. 1991. A low molecular weight allergen of white birch (Betula verrucosa) is highly homologous to human profilin[J]. International Archives of Allergy & Applied Immunology, 94(1-4):368-370. Valenta R, Ferreira F, Grote M, et al. 1993. Identification of profilin as an actin-binding protein in higher plants[J]. Journal of Biological Chemistry, 268(30):22777-22781. Wallar B J, Alberts A S. 2003. The formins: active scaffolds that remodel the cytoskeleton[J]. Trends in Cell Biology, 13(8):435-446. Witke W. 2004. The role of profilin complexes in cell motility and other cellular processes[J]. Trends in Cell Biology, 14(8):461-469. Xu C G, Liu Z T, Zhang L P, et al. 2013. Organization of actin cytoskeleton during meiosis I in a wheat thermo-sensitive genic male sterile line[J]. Protoplasma, 250(1):415-422. Yoo S D, Cho Y H, Sheen J. 2007. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[J]. Nature protocols, 2(7):1565-1572.
