Serpins in plants and green algae

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.     

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data

Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides.     

Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis

Monocotyledons and dicotyledons are distinct, not only in their body plans and developmental patterns, but also in the structural features of their cell walls. The recent completion of the rice (Oryza sativa) genomic sequence and publication of the sequence data, together with the completed database of the Arabidopsis thaliana genome, provide the first opportunity to compare the full complement of cell-wall-related genes from the two distinct classes of flowering plants. We made this comparison by exploiting the fact that Arabidopsis and rice have type I and type II walls, respectively, and therefore represent the two extremes in terms of the structural features of plant cell walls. In this review article, we classify all cell-wall-related genes into 32 gene families, and generate their phylogenetic trees. Using these data, we can phylogenetically compare individual genes of particular interest between Arabidopsis and rice. This comparative genome approach shows that the differences in wall architecture in the two plant groups actually mirror the diversity of the individual gene families involved in the cell-wall dynamics of the respective plant species. This study also identifies putative rice orthologs of genes with well-defined functions in Arabidopsis and other plant species.     

The class III peroxidase multigenic family in rice and its evolution in land plants

Plant peroxidases (class III peroxidases, E.C. 1.11.1.7) are secreted glycoproteins known to be involved in the mechanism of cell elongation, in cell wall construction and differentiation, and in the defense against pathogens. They usually form large multigenic families in angiosperms. The recent completion of rice (Oryza sativa japonica c.v. Nipponbare) genome sequencing allowed drawing up the full inventory of the genes encoding class III peroxidases in this plant. We found 138 peroxidase genes distributed among the 12 rice chromosomes. In contrast to several other gene families studied so far, peroxidase genes are twice as numerous in rice as in Arabidopsis. This large number of genes results from various duplication events that were tentatively traced back using a phylogenetic tree based on the alignment of conserved amino acid sequences. We also searched for peroxidase encoding genes in the major phyla of plant kingdom. In addition to gymnosperms and angiosperms, sequences were found in liverworts, mosses and ferns, but not in unicellular green algae. Two rice and one Arabidopsis peroxidase genes appeared to be rather close to the only known sequence from the liverwort Marchantia polymorpha. The possible relationship of these peroxidases with the putative ancestor of peroxidase genes is discussed, as well as the connection between the development of the class III peroxidase multigenic family and the emergence of the first land plants.     

In search of function for hypothetical proteins encoded by genes of SA-JA pathways in Oryza sativa by in silico comparison and structural modeling

Knowledge of rice genome brings new dimensions to the management of abiotic stresses; however, gene sequences in the rice genome are yet to be assigned structure and function. Hydrogen peroxide, salicylates and jasmonates act as signal molecules in plants employing common machinery to manage abiotic stress. The present work is primarily focused to assign a structurefunction relationship by modeling of the hypothetical proteins of SA-JA signaling pathway known in Arabidopsis thaliana and compare them with corresponding proteins in rice in silico. Thirteen known gene sequences with their encoded proteins for SA/JA pathway in model plant A. thaliana were obtained and similar gene sequences from rice were retrieved at NCBI. Five rice gene sequences Os09g0392100, Os03g0233200, OsJ_33269, OsJ_23610 and Os01g0194300 resulted in hypothetical protein products with unknown structure and function. Modeling and comparison of 5 proteins from rice and Arabidopsis showed 73 - 98% identity with acceptable RMSD values of 0.6 - 1.7 upon superimposition. Results suggest conserved nature of these proteins during evolution. The hypothetical protein from rice contains similar functional protein domain as that in A. thaliana and therefore are likely to perform similar functions in rice. There is a cross talk between the genes in SA/JA pathway wherein Os09g0392100 or EDS1, Os03g0233200 or PR5, OsJ_33269 or PAD4 and OsJ_23610 or SFD-1 activates the pathway and Os01g0194300 or NPR1 inhibit the pathway. Further investigation through wet-lab experiments are in progress to look into suppression/activation of the genes of SAJA signaling in rice plants exposed to abiotic stress.     

Colinearity and Similar Expression Pattern of Rice DREB1s Reveal Their Functional Conservation in the Cold-Responsive Pathway

The clustered genes C-repeat (CRT) binding factor (CBF)1/ dehydration-responsive element binding protein (DREB)1B, CBF2/DREB1C, and CBF3/DREB1A play a central role in cold acclimation and facilitate plant resistance to freezing in Arabidopsis thaliana. Rice (Oryza sativa L.) is very sensitive to low temperatures; enhancing the cold stress tolerance of rice is a key challenge to increasing its yield. In this study, we demonstrate chilling acclimation, a phenomenon similar to Arabidopsis cold acclimation, in rice. To determine whether rice CBF/DREB1 genes participate in this cold-responsive pathway, all putative homologs of Arabidopsis DREB1 genes were filtered from the complete rice genome through a BLASTP search, followed by phylogenetic, colinearity and expression analysis. We thereby identified 10 rice genes as putative DREB1 homologs: nine of these were located in rice genomic regions with some colinearity to the Arabidopsis CBF1–CBF4 region. Expression profiling revealed that six of these genes (Os01g73770, Os02g45450, Os04g48350, Os06g03670, Os09g35010, and Os09g35030) were similarly expressed in response to chilling acclimation and cold stress and were co-expressed with genes involved in cold signalling, suggesting that these DREB1 homologs may be involved in the cold response in rice. The results presented here serve as a prelude towards understanding the function of rice homologs of DREB1 genes in cold-sensitive crops.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.     

A novel bioinformatics approach identifies candidate genes for the synthesis and feruloylation of arabinoxylan

Arabinoxylans (AXs) are major components of graminaceous plant cell walls, including those in the grain and straw of economically important cereals. Despite some recent advances in identifying the genes encoding biosynthetic enzymes for a number of other plant cell wall polysaccharides, the genes encoding enzymes of the final stages of AX synthesis have not been identified. We have therefore adopted a novel bioinformatics approach based on estimation of differential expression of orthologous genes between taxonomic divisions of species. Over 3 million public domain cereal and dicot expressed sequence tags were mapped onto the complete sets of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) genes, respectively. It was assumed that genes in cereals involved in AX biosynthesis would be expressed at high levels and that their orthologs in dicotyledonous plants would be expressed at much lower levels. Considering all rice genes encoding putative glycosyl transferases (GTs) predicted to be integral membrane proteins, genes in the GT43, GT47, and GT61 families emerged as much the strongest candidates. When the search was widened to all other rice or Arabidopsis genes predicted to encode integral membrane proteins, cereal genes in Pfam family PF02458 emerged as candidates for the feruloylation of AX. Our analysis, known activities, and recent findings elsewhere are most consistent with genes in the GT43 families encoding beta-1,4-xylan synthases, genes in the GT47 family encoding xylan alpha-1,2- or alpha-1,3-arabinosyl transferases, and genes in the GT61 family encoding feruloyl-AX beta-1,2-xylosyl transferases.     

A serpin in the cellulosome of the anaerobic fungus Piromyces sp. strain E2

A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homologue, sharing 30 % identity and 50 % similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70 % of known serpins, and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.     

玉米与其他六种植物NBS类抗病基因的进化比较分析

具有核苷酸结合位点(nucleotide binding site,NBS)的抗病基因在植物抵抗各种病原菌侵染中起关键作用。对玉米全基因组中具有NBS结构的基因进行鉴定和分析,并结合水稻、高粱、拟南芥、百脉根、苜蓿和杨树的NBS类基因比较其在数量、复制、染色体定位和亲缘关系上的进化差异。发现玉米NBS类基因数量、复制数和成簇基因数均明显少于其他植物。低复制频率可能导致玉米NBS类基因较少,并推测可能导致其功能具有多样性。在基因染色体定位上,除高梁外,玉米与其他五种植物相似,呈不均衡分布。此外,进化树分析表明玉米NBS类基因与高粱的亲缘关系最近,与拟南芥的最远,在物种间表现出较高的保守性。结果对掲示玉米NBS基因的进化特点与发掘有益的NBS类抗病基因提供了重要的理论依据。     

Serpin protease inhibitors in plant biology

Protease inhibitors of the serpin family are ubiquitous in the plant kingdom but relatively little is known about their biological functions in comparison with their counterparts in animals. X-ray crystal structures have provided crucial insights into animal serpin functions. The recently solved structure of AtSerpin1 from Arabidopsis thaliana, which has the highly conserved reactive center P2-P1' Leu-Arg-Xaa (Xaa = small residue), displays both conserved and plant-specific serpin features. Sequence homology suggests that AtSerpin1 belongs to serpin Clade B, composed of intracellular mammalian serpins, which is consistent with the lack of strong evidence for secretion of serpins from plant cells. The major in vivo target protease for AtSerpin1 is the papain-like cysteine RD21 protease, a match reminiscent of the inhibition of cathepsins K, L and S by the Clade-B mammalian serpin, SCCA-1 (SERPINB3). The function of AtSerpin1 and other serpins that contain P2-P1' Leu-Arg-Xaa (the 'LR' serpins) in plants remains unknown. However, based on its homology and interactive partners, AtSerpin1 and perhaps other serpins are likely to be involved in regulating programmed cell death or associated processes such as senescence. Abundant accumulation of serpins in seeds and their presence in phloem sap suggest additional functions in plant defense by irreversible inhibition of digestive proteases from pests or pathogens. Here we review the most recent findings in plant serpin biology, focusing on advances in describing the structure and inhibitory specificity of the LR serpins.     

Identification of potential protein regulators bound to the tissue-specific positive and negative cis-acting elements of a green tissue-specific promoter in rice

Although some tissue-specific cis-acting elements have been identified, the molecular mechanisms of tissue-specific gene expression remain elusive. Here, we report the identification by a yeast one-hybrid screen of five proteins, Os10g31330/glycine-rich, Os01g10400/metallothionein-like, Os05g51180/nucleic acid-binding, Os05g37930/unknown and Os01g01689/phosphatidylinositol kinase that bound to either the positive or negative tissue-specific cis elements of a rice promoter from the green tissue-specific D54O gene. These proteins are localised in the nucleus and the genes encoding them are differentially expressed in different tissues, further suggesting their putative roles in regulating gene expression. These results suggest that the green tissue-specific expression of the D54O gene may be regulated by the interaction of multiple proteins with cis elements in the promoter region.     

家蚕微孢子虫丝氨酸蛋白酶抑制剂蛋白serpin基因NbSPN106的鉴定

摘要：【目的】Serpin在病原与宿主互作中起着重要的作用。本研究旨在分析在家蚕微孢子虫中的丝氨酸蛋白酶抑制剂蛋白（Serpin）的结构特征,原核克隆表达以及Western blotting检测。【方法】 基于家蚕微孢子虫全基因组序列,同源序列比对搜索获得serpin基因序列。利用在线软件分析基因的序列特征,ClustalX对氨基酸序列进行多重序列比对。构建含有GST标签的pGEX4T1-NbSPN106原核重组表达载体,在大肠杆菌BL21（DE3）诱导表达并进行纯化。纯化的重组蛋白免疫小鼠,制备抗体,并与家蚕微孢子虫总蛋白进行Western blotting免疫杂交。【结果】比对搜索在家蚕微孢子虫基因组中发现一个新的serpin基因NbSPN106。NbSPN106蛋白序列长度为384 aa,N端具有信号肽,编码一个42 kDa左右的成熟蛋白。多重序列比对说明NbSPN106具有保守的serpin位点,可能具有抑制功能。免疫杂交在家蚕微孢子虫总蛋白检测到一条45 kDa左右的特异条带。【讨论】生物信息学分析以及免疫杂交结果说明在家蚕微孢子虫中存在NbSPN106。这是在微孢子虫中首次报道发现serpin基因。对研究家蚕微孢子虫与宿主家蚕的互作具有一定的参考价值。     

Identification and characterization of Mini1, a gene regulating rice shoot development

The aerial parts of higher plants are generated from the shoot apical meristem(SAM). In this study, we isolated a small rice(Oryza sativa L.) mutant that showed premature termination of shoot development and was named mini rice 1(mini1). The mutant was first isolated from a japonica cultivar Zhonghua11(ZH11) subjected to ethyl methanesulfonate(EMS)treatment. With bulked segregant analysis(BSA) and map-based cloning method, Mini1 gene was finally fine-mapped to an interval of 48.6 kb on chromosome 9. Sequence analyses revealed a single base substitution from G to A was found in the region, which resulted in an amino acid change from Gly to Asp.The candidate gene Os09g0363900 was predicted to encode a putative adhesion of calyx edges protein ACE(putative HOTHEAD precursor) and genetic complementation experiment confirmed the identity of Mini1. Os09g0363900 contains glucose-methanol-choline(GMC) oxidoreductase and NAD(P)-binding Rossmann-like domain, and exhibits high similarity to Arabidopsis HOTHEAD(HTH). Expression analysis indicated Mini1 was highly expressed in young shoots but lowly in roots and the expression level of most genes involved in auxin biosynthesis and signal transduction were reduced in mutant.We conclude that Mini1 plays an important role in maintaining SAM activity and promoting shoot development in rice.     

Sequence organization and matrix attachment regions of the human serine protease inhibitor gene cluster at 14q32.1

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system for studying the regulation of gene activity and chromatin structure. We demonstrated previously that the six known serpin genes in this region were organized into two subclusters of three genes each that occupied ~370 kb of DNA. To more fully understand the genomic organization of this region, we annotated a 1-Mb sequence contig from data from the Genoscope sequencing consortium ( ). We report that 11 different serpin genes reside within the 14q32.1 cluster, including two novel 1-antiproteinase-like gene sequences, a kallistatin-like sequence, and two recently identified serpins that had not been mapped previously to 14q32.1. The genomic regions proximal and distal to the serpin cluster contain a variety of unrelated gene sequences of diverse function. To gain insight into the chromatin organization of the region, sequences with putative nuclear matrix-binding potential were identified by using the MAR-Wiz algorithm, and these MAR-Wiz candidate sequences were tested for nuclear matrix-binding activity in vitro. Several differences between the MAR-Wiz predictions and the results of biochemical tests were observed. The genomic organization of the serpin gene cluster is discussed. These authors (Stephanie J. Namciu and Richard D. Friedman) contributed equally to this work.     

Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice

Serine hydroxymethyltransferase(SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development,and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, Os SHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller,lethal phenotype under natural ambient CO2 concentrations,but could be restored to wild type with normal growth under elevated CO2levels(0.5% CO2), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to signi fi cant oxidative stress. Also, Os SHM1 was expressed in allorgans tested(root, culm, leaf, and young panicle) but predominantly in leaves. Os SHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the Os SHM1 gene is conserved in rice and Arabidopsis.     

水稻中受体激酶的系统树分析

植物受体激酶(RLKs)在植物细胞内的反应中发挥着重要作用.为了比较拟南芥(Arabidopsis thaliana L.)和水稻(Oryza sativa L.)中受体激酶的进化关系,作者通过对北京华大基因研究中心(BGI)的籼稻蛋白质数据库进行BLASTP搜索,找到267个受体激酶类似基因,根据它们的胞外结构域可以将这些基因分为不同的类型.与拟南芥中受体激酶的系统树比较分析表明,不同类型的受体激酶具有不同的序列保守性,说明在植物进化过程中,不同类型的受体激酶具有不同的进化关系.水稻受体激酶与拟南芥受体激酶BRI1的多序列匹配结果也表明二者可能具有不同的磷酸化位点.