LeSBT1, a subtilase from tomato plants. Overexpression in insect cells, purification, and characterization

The cDNA of a tomato subtilase designated LeSBT1 was cloned from a tomato flower cDNA library. The deduced amino acid sequence indicated for LeSBT1 the structure of a prepro-protein targeted to the secretory pathway by virtue of an amino-terminal signal peptide. LeSBT1 was expressed in the baculovirus/insect cell system and a processed 73-kDa form of LeSBT1, lacking both signal peptide and prodomain, was purified to homogeneity from culture supernatants. This 73-kDa LeSBT1, however, lacked proteolytic activity. Zymogen activation to yield 68-kDa LeSBT1 required the additional processing of an amino-terminal autoinhibitory peptide in a strictly pH-dependent manner. Mature 68-kDa LeSBT1 showed highest activity at acidic pH consistent with its presumed localization in the apoplast of the plant cell. In comparison to other plant subtilases, LeSBT1 exhibited a narrower substrate specificity in that it cleaves only polypeptide substrates preferentially but not exclusively carboxyl-terminal of glutamine residues. The possible involvement of LeSBT1 in selective proprotein processing is discussed with reference to the related mammalian proprotein convertases.     

Applicability of multigene family-specific antibodies toward studies of the subtilases in Arabidopsis thaliana

Polyclonal antibodies were made to two synthetic peptides with sequences patterned after conserved regions in a multigene family of 56 subtilisin-related proteolytic enzymes in Arabidopsis thaliana. GST-fusion proteins encompassing full-length or partial cDNAs bearing putative epitope regions were cloned and expressed in Escherichia coli. Immunoblots showed that the antibodies bound 12 of 13 fusion proteins tested. About 27 more subtilase genes code for regions with sequences very similar to the epitope regions of the subtilases that were visualized on the immunoblots. Subtilases in rosette and cauline leaves, stems, flowers, and siliques could be distinguished by the antibodies; some binding the two antibodies to similar extents, while others bind preferentially or almost exclusively to one or the other antibody. When antibodies were used to monitor ion-exchange fractionation of seedling extracts, one specific subtilase was identified by LC-MS-MS. The specificity of the antibodies extended to subtilases in soybean. These studies show that multigene family-specific antibodies can be applied to the study of gene families, where sequence similarities make it difficult to produce antibodies specific for each individual protein in the group.     

Subtilase Genes Diversity in the Biogas Digester Microbiota

Biogas digesters contain microbial assemblages that process a mass of extracellular polymeric substances from animal manure and domestic wastewater; however, due to the limitation of available technology in cultivation of majority of the micro-organisms in biogas digesters, the enzymatic potential of these microbial communities remains largely unexplored. In this study, to evaluate subtilase gene diversity in a biogas digester, the partial sequences of the gene were directly amplified from the metagenomic DNA by using consensus-degenerate primers. The desired PCR products were cloned into pGEM-T Easy vector, and thirty positive clones were chose for Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis, from which thirteen distinguished patterns were obtained and then sequenced. Phylogenetic analysis showed that ten out of the thirteen sequences were related to the subtilase genes in GenBank and were grouped into three families of the subtilases superfamily. The nucleotide sequences analysis through BLAST search revealed that none of the partial genes the authors isolated showed significant similarity against the non-redundant Nucleotide database of NCBI. Meanwhile, the deduced amino acid sequences of ten partial subtilase genes showed moderate identities to the previously identified sequences in GenBank, with a range from 39 to 61%. Collectively, the data indicate that there is a great diversity of subtilase genes in the biogas digester; and may be a rich reservoir for novel subtilase genes.     

Dramatic Number Variation of R Genes in Solanaceae Species Accounted for by a Few R Gene Subfamilies

Most disease resistance genes encode nucleotide-binding-site (NBS) and leucine-rich-repeat (LRR) domains, and the NBS-LRR encoding genes are often referred to as R genes. Using newly developed approach, 478, 485, 1,194, 1,665, 2,042 and 374 R genes were identified from the genomes of tomato Heinz1706, wild tomato LA716, potato DM1-3, pepper Zunla-1 and wild pepper Chiltepin and tobacco TN90, respectively. The majority of R genes from Solanaceae were grouped into 87 subfamilies, including 16 TIR-NBS-LRR (TNL) and 71 non-TNL subfamilies. Each subfamily was annotated manually, including identification of intron/exon structure and intron phase. Interestingly, TNL subfamilies have similar intron phase patterns, while the non-TNL subfamilies have diverse intron phase due to frequent gain of introns. Prevalent presence/absence polymorphic R gene loci were found among Solanaceae species, and an integrated map with 427 R loci was constructed. The pepper genome (2,042 in Chiltepin) has at least four times of R genes as in tomato (478 in Heinz1706). The high number of R genes in pepper genome is due to the amplification of R genes in a few subfamilies, such as the Rpi-blb2 and BS2 subfamilies. The mechanism underlying the variation of R gene number among different plant genomes is discussed.     

Genome-wide and molecular evolution analysis of the subtilase gene family in Vitis vinifera

Background

Vitis vinifera (grape) is one of the most economically significant fruit crops in the world. The availability of the recently released grape genome sequence offers an opportunity to identify and analyze some important gene families in this species. Subtilases are a group of subtilisin-like serine proteases that are involved in many biological processes in plants. However, no comprehensive study incorporating phylogeny, chromosomal location and gene duplication, gene organization, functional divergence, selective pressure and expression profiling has been reported so far for the grape.

Results

In the present study, a comprehensive analysis of the subtilase gene family in V. vinifera was performed. Eighty subtilase genes were identified. Phylogenetic analyses indicated that these subtilase genes comprised eight groups. The gene organization is considerably conserved among the groups. Distribution of the subtilase genes is non-random across the chromosomes. A high proportion of these genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the subtilase gene family. Analyses of divergence and adaptive evolution show that while purifying selection may have been the main force driving the evolution of grape subtilases, some of the critical sites responsible for the divergence may have been under positive selection. Further analyses of real-time PCR data suggested that many subtilase genes might be important in the stress response and functional development of plants.

Conclusions

Tandem duplications as well as purifying and positive selections have contributed to the functional divergence of subtilase genes in V. vinifera. The data may contribute to a better understanding of the grape subtilase gene family.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1116) contains supplementary material, which is available to authorized users.     

Structural features of plant subtilases

Serine proteases of the subtilase family are present in Archaea, Bacteria and Eukarya. Many more subtilases are found in plants as compared to other organisms, implying adaptive significance for the expansion of the subtilase gene family in plants. Structural data, however, were hitherto available only for non-plant subtilases. We recently solved the first structure of a plant subtilase, SlSBT3 from tomato (Solanum lycopersicum). SlSBT3 is a multidomain enzyme displaying a subtilisin, a Protease-Associated (PA) and a fibronectin (Fn) III-like domain. Two prominent features set SlSBT3 apart from other structurally elucidated subtilases: (i) activation by PA domain-mediated homo-dimerization and (ii) calcium-independent activity and thermostability. To address the question whether these characteristics are unique features of SlSBT3, or else, general properties of plant subtilases, homology models were calculated for representative proteases from tomato and Arabidopsis using the SlSBT3 structure as template. We found the major structural elements required for the stabilization of the subtilisin domain to be conserved among all enzymes analyzed. PA domain-mediated dimerization as an auto-regulatory mechanism of enzyme activation, on the other hand, appears to be operating in only a subset of the analyzed subtilasesKey words: Arabidopsis thaliana, enzyme structure, homology modeling, proprotein convertase, protease-associated domain, proteolysis, Solanum lycopersicum, SBT3, subtilisin, thermostabilitySubtilases constitute the second largest family of serine peptidases, both in terms of number of sequences and characterized enzymes (merops.sanger.ac.uk/).¹ The function of subtilases ranges from the non-selective degradation of proteins by e.g., subtilisin Carlsberg in Bacillus licheniformis, to the highly specific maturation of peptide hormones and processing of protein precursors by e.g., kexin in Saccharomyces cerevisiae and proprotein convertases in mammals. In higher plants, subtilases are represented by large gene families comprising 56 members in Arabidopsis thaliana and 63 in rice.^2,3 The expansion of the subtilase family in plants as compared to animals has apparently been accompanied by the acquisition of novel physiological roles that are plant-specific. Plant subtilases were shown to be involved in stomata and seed development,^4,5 in the maintenance of the shoot apical meristem and the cell wall,^6,7 in the processing of peptide growth factors,^8,9 and in responses to the biotic and abiotic environment.^10,11 To address the question whether the adoption of specific roles in plant physiology is reflected in unique structural or biochemical features that distinguish subtilases in plants from those in other organisms, we recently characterized the subtilase SlSBT3 from tomato¹² and solved its structure by X-ray crystallography.^13,14SlSBT3 is an extracellular 79 kDa glycoprotein that exhibits a remarkable level of stability at elevated temperatures and alkaline pH.¹² Like most other subtilases, SlSBT3 is synthesized as a pre-pro-protein and targeted for secretion by an N-terminal signal peptide. Maturation of SlSBT3 involves cleavage of its prodomain, which is a prerequisite for passage through the secretory pathway.¹² Mature SlSBT3 features a protease-associated (PA) domain as a large insertion between the His and Ser active site residues of the protease domain and a fibronectin (Fn) III-like domain as C-terminal extension.¹⁴ This domain architecture (Fig. 1A) is shared with the majority of plant subtilases (e.g., 54 of the 56 subtilases in Arabidopsis). Unlike other structurally elucidated subtilases from bacteria, fungi and animals, SlSBT3 was found to be free of Ca²⁺ in its native state and independent of Ca²⁺ with respect to activity and thermostability. The ability of SlSBT3 to form homodimers is also unique and appears to be critical for enzyme activity and stability.¹⁴ For this addendum, we calculated homology models for representative subtilases from Arabidopsis and tomato to investigate whether PA domain-mediated homo-dimerization and calcium-independent thermostability are unique features of the SlSBT3 structure, or whether they can serve as a first paradigm for the structural biology of plant subtilases in general.Open in a separate windowFigure 1Structural comparison of plant subtilases. (A) Domain architecture of SlSBT3. In addition to the domain borders the three residues constituting the active site are displayed. (B) Structure of the SlSBT3 monomer. Color coding of the domains is like in (A). The bound chloromethylketone (cmk)-inhibitor is shown as ball model in yellow (carbon), red (oxygen) and blue (nitrogen). (C) Functional homodimer of SlSBT3. The region of the direct contact between the two PA domains (gold, purple) is highlighted. In this and all the following panels, a sequence alignment of the relevant regions in SlSBT3 and the modeled subtilases is shown on the right. The sequences highlighted in color were included in the structural alignment on the left. (D) Structure of the partially conserved β-hairpin. (E) Structure of the region corresponding to the conserved calcium-binding site 1 (Ca-1) in thermitase (yellow sticks, PDB code: 1THM). (F) Functional substitution of the conserved Ca-2 (white sticks, PDB code 1S2N) site by a lysine side chain in plant subtilases. (G) Structure of the region corresponding to the less conserved Ca-3 site in thermitase (yellow sticks, PDB code: 1THM). For details, see text.     

The phytochrome gene family in tomato and the rapid differential evolution of this family in angiosperms

A reexamination of the genome of the tomato (renamed Solanum lycopersicum L.) indicates that it contains five, or at most perhaps six, phytochrome genes (PHY), each encoding a different apoprotein (PHY). Five previously identified tomato PHY genes have been designated PHYA, PHYB1, PHYB2, PHYE, and PHYF. A molecular phylogenetic analysis is consistent with the hypothesis that the angiosperm PHY family is composed of four subfamilies (A, B, C/F, and E). Southern analyses indicate that the tomato genome does not contain both a PHYC and a PHYF. Molecular phylogenetic analyses presented here, which utilize for the first time full-length PHY sequences from two completely characterized angiosperm gene families, indicate that tomato PHYF is probably an ortholog of Arabidopsis PHYC. They also confirm that the angiosperm PHY family is undergoing relatively rapid differential evolution. Assuming PHYF is an ortholog of PHYC, PHY genes in eudicots are evolving (Ka/site) at 1.52-2.79 times the rate calculated as average for other plant nuclear genes. Again assuming PHYF is an ortholog of PHYC, the rate of evolution of the C and E subfamilies is at least 1.33 times the rate of the A and B subfamilies. PHYA and PHYB in eudicots are evolving at least 1.45 times as fast as their counterparts in the Poaceae. PHY functional domains also exhibit different evolutionary rates. The C-terminal region of angiosperm PHY (codons 800-1105) is evolving at least 2.11 times as fast as the photosensory domain (codons 200-500). The central region of a domain essential for phytochrome signal transduction (codons 652-712) is also evolving rapidly. Nonsynonymous substitutions occur in this region at 2.03-3.75 times the average rate for plant nuclear genes. It is not known if this rapid evolution results from selective pressure or from the absence of evolutionary constraint.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

Autolysis of a novel multidomain subtilase-cold-adapted deseasin MCP-01 is pH-dependent and the surface loops in its catalytic domain, the linker, and the P_proprotein domain are susceptible to proteolytic attack

Cold-adapted deseasin MCP-01 is a novel type subtilase with a multidomain structure containing a catalytic domain, a linker, a P_proprotein domain, and a PKD domain. Its autolysis was pH-dependent due to its flexible structure. N-terminal sequence analysis of the autolytic peptides revealed four autolytic sites in the catalytic domain. Three of these are in the same loops as mesophilic subtilases and one is unlike anything previously reported. Two autolytic sites were deduced in its linker and three in its P_proprotein domain, indicating the linker and the P_proprotein domain are flexible and susceptible to proteolytic attacks. Therefore, during MCP-01 autolysis, the linker and the P_proprotein domain of MCP-01 were easily attacked by proteolysis, resulting in cleavage of the C-terminal region. At the same time, some autolytic sites in the surface loops of the catalytic domain were cleaved. This is the first report describing the autolytic mechanism of a multidomain subtilase.     

Disruption of the subtilase gene, albin1, in Ophiostoma piliferum

Wood sapstaining fungi produce multiple proteases that break down wood protein. Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B. Hoffman and C. Breuil, Curr. Genet. 41:168-175, 2002). In this work we examined the role of the subtilase Albin1 from Ophiostoma piliferum. Reamplification of cDNA ends PCR was used to obtain the albin1 gene sequence. The encoded subtilase is probably extracellular and involved in nutrient acquisition. This gene was disrupted with an Agrobacterium tumefaciens-mediated transformation system. Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood. Thus, albin1 plays an important role in O. piliferum's ability to acquire nitrogen from wood proteins.     

番茄PPR基因家族的鉴定与生物信息学分析

PPR(Pentatricopeptide repeats)基因家族在植物中广泛存在, 其在植物生长发育过程中至关重要。文章采用生物信息学方法, 利用Pfam已鉴定的PPR保守结构域序列检索番茄(Solanum lycopersicum L.)基因组计划注释的蛋白序列, 最终确定了番茄中可能存在的471个PPR编码基因; 根据拟南芥(Arabidopsis thaliana L.)中鉴定的各个结构域的特点对其进行了蛋白结构分析、分类和保守序列分析, 并对番茄PPR基因家族进行了系统进化树构建、染色体定位、亚细胞定位预测、表达和GO分析等。结果表明：番茄PPR基因家族分为P和PLS两个亚家族, 各占序列数目的一半, PLS亚家族又分为PLS、E、E+和DYW四类, 且在进化树中形成不同的分支; 各个结构域在植物中非常保守; PPR基因家族分布在番茄12条染色体上, 且多数无内含子结构; 大部分PPR蛋白具有线粒体或叶绿体定位序列, GO分析表明PPR蛋白参与RNA相关的生物学过程     

Identification and chromosomal location of four subfamilies of the rubisco small subunit genes in common wheat

Summary Three different 3 noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3 noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5 region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.     

番茄热激蛋白90的全基因组鉴定及分析

热激蛋白90(Heat shock protein 90,Hsp90)是植物应对不良环境胁迫产生的一类特定的抗逆蛋白。文章以番茄(Solanum lycopersicum L.)基因组数据为平台,借助生物信息学方法对Hsp90基因家族进行鉴定与分析。结果表明,番茄至少含有7个Hsp90基因,不均匀分布在6条染色体上,氨基酸序列长度为267~794aa,内含子数目为2~19;共线性分析发现两对基因(Hsp90-1和Hsp90-3,Hsp90-5和Hsp90-7)以片段重复形式存在。MEME(Multiple Em for Motif Elicitation)分析显示,番茄Hsp90基因编码的氨基酸序列具有多个保守基序;聚类分析揭示番茄、水稻(Oryza sativa L.)和拟南芥(Arabidopsis thaliana L.)Hsp90基因可以分为5组,存在3对直系同源基因和4对旁系同源基因;基于RNA-seq数据库表达分析发现,3个基因(Hsp90-5、Hsp90-6和Hsp90-7)在营养器官和生殖器官中表达量较高,4个基因(Hsp90-1、Hsp90-2、Hsp90-3和Hsp90-4)除在番茄转色后10 d的果实中表达量较高外,其余组织中表达量均较低;对Hsp90基因启动子序列进行分析,发现了多个参与植物对逆境胁迫的顺式作用元件,如HSE、CCAAT-box。此外,qRT-PCR检测结果表明,在叶片热胁迫条件下,番茄Hsp90基因的表达量均存在增强趋势,表明这些基因参与了番茄叶片应对高温胁迫的反应。研究结果为鉴定番茄Hsp90基因的功能和进化起源奠定了基础。