The PR-1a promoter contains a number of elements that bind GT-1-like nuclear factors with different affinity

A novel cDNA encoding for a peptidyl-prolyl-cis-trans-isomerase (PPIase) belonging to the FK506-binding protein (FKBP) family was isolated from wheat. It contains an open reading frame of 559 amino acids and it represents the first plant FKBP-PPIase to be cloned. It possesses a unique sequence which is composed of three FKPB-like domains, in addition to a putative tetratricopeptide repeat (TPR) motif and a calmodulin-binding site. The recombinant FKBP-PPIase expressed in and purified from Escherichia coli exhibits PPIase activity that is efficiently inhibited by the immunosuppressive drugs FK506 and rapamycin. Northern blot analysis showed that wheat FKBP was found mainly in young tissues. Polyclonal antibodies revealed the presence of cross-reacting proteins in embryos, roots and shoots. The unique structural features, the enzymatic activity and the presence of putative isoforms in wheat tissues indicate the possibility of the involvement of wheat PPIase in essential biological functions, similar to other members of the FKBP gene family.     

Molecular cloning and characterization of genes encoding FK506-binding proteins (FKBPs) in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The family of FK506-binding proteins (FKBPs) consists of several members, which show peptidyl prolyl cis–trans isomerase (PPIase) activity. PPIases facilitate the conversion of peptidyl prolyl bonds from cis to trans conformation, a rate-limiting step in protein folding. In the present study, we carried out cloning of cDNAs encoding three different wheat FKBPs viz., TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1. In silico analysis suggested their likely localization to nucleus, cytosol and endoplasmic reticulum, respectively. Biochemical analyses demonstrated that none of the three purified FKBP proteins possesses detectable PPIase activity. Several putative interacting partners of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1were identified using online software tools. The results of this study provide further evidence that PPIase activity in plant FKBPs is not conserved, and these proteins may be playing important roles in the cell through interaction with target proteins.     

Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.     

普通小麦中一氧化氮相关因子(TaNOA)编码基因的克隆和分子生物学分析

一氧化氮是动植物体内重要的信号分子。本研究利用同源克隆技术从六倍体普通小麦中获得一个一氧化氮相关因子(TaNOA)编码基因的全长基因组和cDNA克隆。该基因具有13个外显子和12个内含子,与拟南芥以及水稻中同源基因结构相似。根据cDNA推导的氨基酸序列与拟南芥AtNOA1的序列一致性达60%以上,具备P-环GTPaseG4-G5-G1-G2-G3的排列特征和保守的序列。对其中2个内含子的测序分析表明在六倍体小麦中TaNOA至少有3个成员。进一步用中国春小麦缺体-四体材料将这3个TaNOA基因成员分别定位在第六同源群的6A、6B和6D染色体上,本研究中获得的成员定位于6B染色体上,因此将其命名为TaNOA-B1。原生质体表达实验表明,TaNOA-B1可能定位在线粒体中。TaNOA基因在小麦根、叶片中表达较高,在幼穗和小花中有少量表达,茎中几乎检测不到表达。TaNOA的转录本水平还因脱落酸或盐处理而上升,表明它可能参与小麦对非生物胁迫的反应。本研究为进一步克隆六倍体小麦中TaNOA的其他成员及研究该基因在小麦中的功能奠定了基础。     

Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.     

Genome-wide analysis and environmental response profiling of the FK506-binding protein gene family in maize (Zea mays L.)

The FK506-binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, and have been implicated in a wide spectrum of biological processes, including protein folding, hormone signaling, plant growth, and stress responses. Genome-wide structural and evolutionary analyses of the entire FKBP gene family have been conducted in Arabidopsis and rice. In the present study, a genome-wide analysis was performed to identify all maize FKBP genes. The availability of complete maize genome sequences allowed for the identification of 24 FKBP genes. Chromosomal locations in the maize genome were determined and the protein domain and motif organization of ZmFKBPs analyzed. The phylogenetic relationships between maize FKBPs were also assessed. The expression profiles of ZmFKBP genes were measured under different environmental conditions and revealed distinct ZmFKBP gene expression patterns under heat, cold, salt, and drought stress. These data not only contribute to a better understanding of the complex regulation of the maize FKBP gene family, but also provide evidence supporting the role of FKBPs in multiple signaling pathways involved in stress responses. This investigation may provide valuable information for further research on stress tolerance in plants and potential strategies for enhancing maize survival under stressful conditions.     

A novel role for the immunophilin FKBP52 in copper transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.     

PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

Structure and expression ofOsMRE11 in rice

In yeast and human cells, the Mre11 complex, which consists of Mre11, Rad50, and Xrs2/Nbs1 proteins, participates in basic aspects of chromosome metabolism, such as the repair of meiotic DNA breaks and telomere maintenance. In this study, we isolated a full-length cDNA clone, pOsMrell, encoding a rice ortholog of the Mre11 protein. Its predicted protein sequence (M_r = 79.2 kDa and pl value = 5.91) contains a metallo-phosphoesterase domain at its N-terminal region, and a single putative DNA binding domain in the central region of the protein, with significant homology to corresponding motifs in human and yeast Mre11 proteins. TheOsMRE11 gene is constitutively expressed in all tissues examined here, including leaves, roots, tillers, and meristems, as well as in undifferentiated callus cells. When 10-d-old rice seedlings were treated with 0.025% methyl methanesulfonate (MMS) or 30 watts of UV-C light, they were apparently damaged by those genotoxic agents, with plants being more seriously injured by the latter. RNA gel blot analysis showed that the level ofOsMRE11 mRNA remained unchanged during the 1- to 4-d incubation period with MMS. In contrast,OsMRE11 expression appeared to increase after 3 d of irradiation. In addition, treatments with salicylic acid and jasmonic acid, two important defense-related hormones, significantly activated theOsMRE11 gene. Based on these results, we discuss the possible functions of the OsMre11 protein in a mechanism by which the stability of rice chromosomes is maintained.