新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

Development of Full-Length cDNAs from Chinese Cabbage (Brassica rapa Subsp. pekinensis) and Identification of Marker Genes for Defence Response

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species

Programmed cell death (PCD) is of fundamental importance to development and defense in animals and plants. In plants, a well-recognized form of PCD is hypersensitive response (HR) triggered by pathogens, which involves the generation of reactive oxygen species (ROS) and other signaling molecules. While the mitochondrion is a master regulator of PCD in animals, the chloroplast is known to regulate PCD in plants. Arabidopsis Mosaic Death 1 (MOD1), an enoyl-acyl carrier protein (ACP) reductase essential for fatty acid biosynthesis in chloroplasts, negatively regulates PCD in Arabidopsis. Here we report that PCD in mod1 results from accumulated ROS and can be suppressed by mutations in mitochondrial complex I components, and that the suppression is confirmed by pharmaceutical inhibition of the complex I-generated ROS. We further show that intact mitochondria are required for full HR and optimum disease resistance to the Pseudomonas syringae bacteria. These findings strongly indicate that the ROS generated in the electron transport chain in mitochondria plays a key role in triggering plant PCD and highlight an important role of the communication between chloroplast and mitochondrion in the control of PCD in plants.     

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsible for the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsis seedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, and this was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assays revealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependent and competitive manner. NAE 12:0 was 10-20 times more potent an inhibitor of LOX activities than lauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but not FFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting that NAE 12:0 may be used in planta to manipulate oxylipin metabolism.     

Significant involvement of PEP-CK in carbon assimilation of C4 eudicots

Background and Aims

C₄ eudicot species are classified into biochemical sub-types of C₄ photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C₄ eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C₄ photosynthetic enzymes.

Methods

Three C₄ species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C₄ enzymes were measured for biochemical characteristics.

Key Results

Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity.

Conclusions

Involvement of PEP-CK in C₄ NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C₄ Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C₄ eudicots.     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.     

Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine

Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2–2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2–2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.     

Effector-Triggered Immune Response in Arabidopsis thaliana Is a Quantitative Trait

We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors.     

Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthora melonis, Phytophthora capsici and water immersion in disease resistant and susceptible cucumber cultivars

L-type lectin receptor kinase (LecRK) proteins are an important family involved in diverse biological processes such as pollen development, senescence, wounding, salinity and especially in innate immunity in model plants such as Arabidopsis and tobacco. Till date, LecRK proteins or genes of cucumber have not been reported. In this study, a total of 25 LecRK genes were identified in the cucumber genome, unequally distributed across its seven chromosomes. According to similarity comparison of their encoded proteins, the Cucumis sativus LecRK (CsLecRK) genes were classified into six major clades (from Clade I to CladeVI). Expression of CsLecRK genes were tested using QRT-PCR method and the results showed that 25 CsLecRK genes exhibited different responses to abiotic (water immersion) and biotic (Phytophthora melonis and Phytophthora capsici inoculation) stresses, as well as that between disease resistant cultivar (JSH) and disease susceptible cultivar (B80). Among the 25 CsLecRK genes, we found CsLecRK6.1 was especially induced by P. melonis and P. capsici in JSH plants. All these results suggested that CsLecRK genes may play important roles in biotic and abiotic stresses.     

Alleviation of Salt Stress by Enterobacter sp. EJ01 in Tomato and Arabidopsis Is Accompanied by Up-Regulation of Conserved Salinity Responsive Factors in Plants

Microbiota in the niches of the rhizosphere zones can affect plant growth and responses to environmental stress conditions via mutualistic interactions with host plants. Specifically, some beneficial bacteria, collectively referred to as Plant Growth Promoting Rhizobacteria (PGPRs), increase plant biomass and innate immunity potential. Here, we report that Enterobacter sp. EJ01, a bacterium isolated from sea china pink (Dianthus japonicus thunb) in reclaimed land of Gyehwa-do in Korea, improved the vegetative growth and alleviated salt stress in tomato and Arabidopsis. EJ01 was capable of producing 1-aminocy-clopropane-1-carboxylate (ACC) deaminase and also exhibited indole-3-acetic acid (IAA) production. The isolate EJ01 conferred increases in fresh weight, dry weight, and plant height of tomato and Arabidopsis under both normal and high salinity conditions. At the molecular level, short-term treatment with EJ01 increased the expression of salt stress responsive genes such as DREB2b, RD29A, RD29B, and RAB18 in Arabidopsis. The expression of proline biosynthetic genes (i.e. P5CS1 and P5CS2) and of genes related to priming processes (i.e. MPK3 and MPK6) were also up-regulated. In addition, reactive oxygen species scavenging activities were enhanced in tomatoes treated with EJ01 in stressed conditions. GFP-tagged EJ01 displayed colonization in the rhizosphere and endosphere in the roots of Arabidopsis. In conclusion, the newly isolated Enterobacter sp. EJ01 is a likely PGPR and alleviates salt stress in host plants through multiple mechanisms, including the rapid up-regulation of conserved plant salt stress responsive signaling pathways.     

Potential but limited redundant roles of MtPIN4, MtPIN5 and MtPIN10/SLM1 in the development of Medicago truncatula

Auxin polar transport is crucial in regulating plant growth and patterning. As auxin efflux carriers, the PIN FORMED (PIN) proteins are responsible for transportation of auxin out of the cell. There are eight and ten PIN members in Arabidopsis (AtPIN) and Medicago truncatula (MtPIN), respectively. Compared with MtPIN10/SMOOTH LEAF MARGIN1 (SLM1), MtPIN4 exhibits a closer relationship with AtPIN1 based phylogenetic analysis. In addition, the gene structure and distribution of transmembrane segments of MtPIN4, MtPIN5 and MtPIN10/SLM1 are similar, implying possible redundant roles among them. However, analysis using Gene Expression Atlas revealed different expression patterns among MtPIN4, MtPIN5 and MtPIN10/SLM1. Loss of function of MtPIN10/SLM1 in M. truncatula resulted in pleiotropic phenotypes in different organs, which are similar with the defects in the pin1 mutant of Arabidopsis, suggesting that the MtPIN10/SLM1 is a putative ortholog of AtPIN1. MtPIN4, MtPIN5 and MtPIN10/SLM1 may have limited redundant functions in the development of M. truncatula. The creation of double and triple mutants will help to elucidate their potential roles in auxin transport and plant development.