Comparison of Cytochrome P450 Genes from Six Plant Genomes

Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms, CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families, of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387 P450 sequences have been identified and annotated in plant databases. As no new angiosperm CYP families have been discovered since 2004, it is now apparent that the sampling of CYP diversity is beginning to plateau. This review presents a comparison of 1,415 cytochrome P450 sequences from the six sequenced genomes of Vitis vinifera (grape), Carica papaya (papaya), Populus trichocarpa (poplar), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis or mouse ear’s cress) and Physcomitrella patens (moss). An evolutionary analysis is presented that tracks land plant P450 innovation over time from the most ancient and conserved sequences to the newest dicot-specific families. The earliest or oldest P450 families are devoted to the essential biochemistries of sterol and carotenoid synthesis. The next evolutionary radiation of P450 families appears to mediate crucial adaptations to a land environment. And, the newest CYP families appear to have driven the diversity of angiosperms in mediating the synthesis of pigments, odorants, flavors and order-/genus-specific secondary metabolites. Family-by-family comparisons allow the visualization of plant genome plasticity by whole genome duplications and massive gene family expansions via tandem duplications. Molecular evidence of human domestication is quite apparent in the repeated P450 gene duplications occurring in the grape genome.     

Cloning and characterization of two cytochrome P450 CYP6AX1 and CYP6AY1 cDNAs from Nilaparvata lugens Stål (Homoptera: Delphacidae)

Two full‐length P450 cDNAs, CYP6AX1 and CYP6AY1, were cloned from the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Both CYP6AX1 and CYP6AY1 are typical microsomal P450s and their deduced amino acid sequences share common characteristics with other members of the insect P450 CYP6 family. CYP6AX1 and CYP6AY1 show the highest percent identity (36%) of amino acid to each other; both of them have 31–33% amino acid identity with CYP6B1 from Papilio polyxenes (Lepidoptera: Papilionidae), CYP6B4 from Papilio glaucus (Lepidoptera: Papilionidae), and CYP6B8 from Helicoverpa zea (Lepidoptera: Noctuidae). Phylogenetic analysis showed the clustering of CYP6AX1 and CYP6AY1 was in the clade including CYP6AE1 from Depressaria pastinacella (Lepidoptera: Oecophoridae) and the CYP6B family members from Helicoverpa and Papilio species. Northern blot analysis revealed that both of the P450s were induced by the resistant rice variety B5 (Oryza sativa L), and CYP6AY1 was expressed at a higher level than CYP6AX1. The results suggest that more than one P450s are likely involved in metabolism of rice allelochemicals and that they are possibly important components in adaptation of Nilaparvata lugens to host rice. Arch. Insect Biochem. Physiol. 64:88–99, 2007. © 2007 Wiley‐Liss, Inc.     

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila : evidence for involvement in host plant utilization

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3^′ RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs. Received: 14 August 1997 / Accepted: 24 March 1998     

Trends in comparative genetics and their potential impacts on wheat and barley research

We review some general points about comparative mapping, the evolution of gene families and recent advances in the understanding of angiosperm phylogeny. These are considered in relation to studies of large-genome cereals, particularly barley (Hordeum vulgare) and wheat (Triticum aestivum), with reference to methods of gene isolation. The relative merits of direct map-based cloning in barley and wheat, utilization of the smaller genome of rice (Oryza sativa) and gene homology methods that utilize information from model species such as Arabidopsis thaliana are briefly discussed.     

Tissue-specific expression of rice CYP72A21 induced by auxins and herbicides

Cytochrome monooxygenase P450s (CYPs) comprise one of the largest enzyme families in plants. Some P450s are involved in xenobiotic metabolism: they confer herbicide tolerance and are induced by chemical treatments. We isolated a novel P450 cDNA, CYP72A21 (accession number, AB237166), from rice (Oryza sativa L. cv. Nipponbare) seedlings treated with a mixture of 2,4-dichlorophenoxyacetic acid (2,4-D), chlorotoluron, phenobarbital, salicylic acid, and naphthalic anhydride (each 100 μM). We also isolated the gene’s promoter region. Endogenous CYP72A21 expression in rice seedlings treated with 2,4-D, herbicides esprocarb, or trifluralin was increased in the aerial part of seedlings. An expression plasmid, pI21pg, containing the GUS gene under the control of the CYP72A21 promoter was introduced into rice plants. GUS was expressed constitutively in roots, but this expression was suppressed by 2,4-D treatment. 2,4-D and other auxins induced GUS expression effectively in the stem and leaves. Histological observation revealed that GUS was expressed mainly in the base of the stem. Treatment with the herbicides acetochlor, esprocarb, and propyzamide induced GUS expression in the aerial parts of the seedlings. The CYP72A21 promoter was highly responsive to treatments with various chemicals, and thus might be useful for producing transgenic plants for biomonitoring of environmental chemicals.     

Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot

Data mining methods have been used to identify 356 Cyt P450 genes and 99 related pseudogenes in the rice (Oryza sativa) genome using sequence information available from both the indica and japonica strains. Because neither of these genomes is completely available, some genes have been identified in only one strain, and 28 genes remain incomplete. Comparison of these rice genes with the 246 P450 genes and 26 pseudogenes in the Arabidopsis genome has indicated that most of the known plant P450 families existed before the monocot-dicot divergence that occurred approximately 200 million years ago. Comparative analysis of P450s in the Pinus expressed sequence tag collections has identified P450 families that predated the separation of gymnosperms and flowering plants. Complete mapping of all available plant P450s onto the Deep Green consensus plant phylogeny highlights certain lineage-specific families maintained (CYP80 in Ranunculales) and lineage-specific families lost (CYP92 in Arabidopsis) in the course of evolution.     

Analysis of Substrate Specificity of Pig CYP2B22 and CYP2C49 towards Herbicides by Transgenic Rice Plants

We introduced two novel types of pig (Sus scrofa) cytochrome P450, CYP2B22 and CYP2C49, into rice plants (Oryza sativa L. cv. ‘Nipponbare’) to produce herbicide-tolerant plants and to confirm the metabolic activities of the cytochrome P450 species. In germination tests, both types of transgenic plants showed tolerance to various herbicides with different modes of action. CYP2B22 rice plants showed tolerance towards 12 herbicides including chlortoluron (100 μM), amiprofos-methyl (2.5 μM), pendimethalin (10 μM), metolachlor (2.5 μM), and esprocarb (20 μM). CYP2C49 rice plants showed tolerance towards 13 herbicides, including chlortoluron (100 μM), norflurazon (0.5 μM), amiprofos-methyl (2.5 μM), alachlor (0.8 μM), and isoxaben (1 μM). The herbicide tolerance was considered to reflect the substrate specificity of the introduced P450 species. We used ¹⁴C-labeled metolachlor and norflurazon to confirm the P450 activity in the transgenic rice plants. The herbicides were metabolized more quickly in the transgenic rice plants than in the nontransgenic rice plants. Therefore, CYP2B22 and CYP2C49 rice plants became more tolerant to various herbicides than nontransgenic control plants because of accelerated metabolism of the herbicides by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, these transgenic rice plants may become useful tools for the breeding of herbicide-tolerant crops.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

Functional complementation of dwf4 mutants of Arabidopsis by overexpression of CYP724A1

An essential step in the biosynthesis of bioactive brassinosteroids (BRs) in plants is the hydroxylation at C-22, a reaction catalyzed by P450 enzymes of the CYP90B and CYP724B subfamilies. Genes for both types of enzymes are present in many species, and in rice (Oryza sativa) and tomato (Solanum lycopersicum) both CYP90B and CYP724B enzymes contribute to C-22 hydroxylation. In Arabidopsis (Arabidopsis thaliana), C-22 hydroxylation of BRs is catalyzed by CYP90B1 (encoded by DWF4) and null dwf4 mutants show severe symptoms of BR-deficiency. CYP724A1 (At5g14400), an Arabidopsis gene of unknown function and limited expression, encodes a P450 sharing less than 55% sequence identity to CYP724B proteins. We used transgenic plants of the null mutants dwf4-102 and a novel allele, bashful (bsf), ectopically expressing the CYP724A1 gene to investigate the potential activity of CYP724A1 as a C-22 hydroxylase of BRs. Defects associated with BR deficiency were reversed and a normal growth habit restored in transgenic dwf4-102 and bsf plants overexpressing CYP724A1. The vegetative phase was prolonged and the transgenic plants were on average larger than wild type plants with respect to several morphometric parameters. Fertility was restored in the transgenic plants but individual siliques yielded fewer and heavier seeds than those of wild type plants. The implications of these findings with regard to the functions of CYP724A1 and the activity of its encoded enzyme are discussed.     

Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids

Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE_2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids.     

The transfer RNA genes in Oryza sativa L. ssp. indica

The availability of the draft genome sequence of Oryza sativa L. ssp. indica has made it possible to study the rice tRNA genes. A total of 596 tRNA genes, including 3 selenocysteine tRNA genes and one suppressor tRNA gene are identified in 127551 rice contigs. There are 45 species of tRNA genes and the revised wobble hypothesis proposed by Guthrie and Abelson is perfectly obeyed. The relationship between codon usage and the number of corresponding tRNA genes is discussed. Redundancy may exist in the present list of tRNA genes and novel ones may be found in the future. A set of 33 tRNA genes is discovered in the complete chloroplast genome of Oryza sativa L. ssp. indica. These tRNA genes are identical to those in ssp. japonica identified by us independently from the origional annotation.     

Analysis of cytochrome P450 genes in silkworm genome (Bombyx mori)

Cytochrome P450 monooxygenases (P450) are im-portant metabolic enzymes involved in the metabolism not only of a wide range of endogenous compounds such as fatty acids, steroids, hormones or vitamins, but also of exogenous substrates such as drugs, chemicals including environmental pollutants, such carcinogens as polycyclic aromatic hydrocarbons, and pesticides[1]. P450s are found virtually in all aerobic organisms, including organisms as diverse as in insects, plants, mammals, birds and bacter…     

Comparative analysis of rice MADS-box genes expressed during flower development

MADS-box genes involved in flower development have been isolated and studied in a wide variety of plant species. However, most of these studies are related to dicot species like Antirrhinum majus, Arabidopsis thaliana and Petunia hybrida. Although the floral structures of typical monocot and dicot flowers differ substantially, previous studies indicate that MADS-box genes controlling floral organ identity in dicots can also be identified in monocot plants like rice and maize. To extend this study further to obtain a more global picture of monocot and dicot MADS-box gene evolution, we performed a phylogenetic study using MADS-box genes from A. thaliana and Oryza sativa. Furthermore, we investigated whether the identified orthologues of Arabidopsis and rice have a conserved expression profile that could indicate conservation of function.     

马尾松细胞分裂素羟化酶基因PmCYP735A克隆与表达分析

该文首次在针叶树种马尾松(Pinus massoniana)中采用cDNA末端快速克隆(RACE)技术克隆,并鉴定出1个CYP735A基因。结果表明:马尾松CYP735A基因(PmCYP735A) cDNA全长为1 744 bp,包括1 647 bp的开放阅读框,44 bp的5'端非翻译区和53 bp的3'端非翻译区。该基因编码蛋白由548个氨基酸残基组成,其二级结构含有丰富的α-螺旋和无规则卷曲。该基因编码蛋白不含跨膜区域,且无信号肽酶切位点,在399～406个和475～484个氨基酸残基存在P450超家族保守特征序列ETLRLYP(ExxRxxP)和血红素结合区域(Heme-binding region) FSFGPRKCVG (FxxGxRxCxG)。系统进化树分析表明,马尾松PmCYP735A与水稻、玉米、拟南芥CYP735A蛋白归属于同一小的进化枝,可可、毛果杨、麻风树和橡胶树等的CYP735A同源蛋白相对集中的定位于另一进化分支。实时定量PCR检测发现,PmCYP735A基因在马尾松根和茎中的表达量显著高于叶,该基因响应外源生长素NAA诱导表达,随着诱导时间呈现先上升再下降的表达趋势。以上结果有助于深入探究CYP735A基因家族的生物学功能及其在物种间的表达调控异同,为进一步挖掘马尾松优异基因资源奠定基础。     

Identification of an active miniature inverted‐repeat transposable element mJing in rice

Miniature inverted‐repeat transposable elements (MITEs) are structurally homogeneous non‐autonomous DNA transposons with high copy numbers that play important roles in genome evolution and diversification. Here, we analyzed the rice high‐tillering dwarf (htd) mutant in an advanced backcross population between cultivated and wild rice, and identified an active MITE named miniature Jing (mJing). The mJing element belongs to the PIF/Harbinger superfamily. japonica rice var. Nipponbare and indica var. 93‐11 harbor 72 and 79 mJing family members, respectively, have undergone multiple rounds of amplification bursts during the evolution of Asian cultivated rice (Oryza sativa L.). A heterologous transposition experiment in Arabidopsis thaliana indicated that the autonomous element Jing is likely to have provides the transposase needed for mJing mobilization. We identified 297 mJing insertion sites and their presence/absence polymorphism among 71 rice samples through targeted high‐throughput sequencing. The results showed that the copy number of mJing varies dramatically among Asian cultivated rice (O. sativa), its wild ancestor (O. rufipogon), and African cultivated rice (O. glaberrima) and that some mJing insertions are subject to directional selection. These findings suggest that the amplification and removal of mJing elements have played an important role in rice genome evolution and species diversification.     

Role of the Rice BAHD Acyltransferase Gene OsAt10 in Plant Cold Stress Tolerance

The rice (Oryza sativa L.) BAHD acyltransferase gene OsAt10 affects growth and metabolism of cells and regulates cell response to environmental stress. However, influence of the OsAt10 gene on low-temperature stress tolerance has not been evaluated in plant cells. Here, cell suspension cultures of plant species Arabidopsis (Arabidopsis thaliana L.), cotton (Gossypium hirsutum L.), white pine (Pinus strobus L.), and rice (Oryza sativa L.) were used to generate transgenic cell lines via Agrobacterium tumefaciens-mediated genetic transformation to examine the effects of OsAt10 on cold stress tolerance. OsAt10 transgenic cell lines of A. thaliana, G. hirsutum, P. strobus, and O. sativa were confirmed by molecular analyses including Southern blotting ND northern blotting, following by physiological and biochemical analyses under cold stress. The experimental results demonstrated that growth rate, cell viability, lipid peroxidation, ion leakage, antioxidative enzyme activity, polyamines level, and cell morphology were changed in transgenic cells under cold stress, compared to the controls. In transgenic A. thaliana cells, overexpression of the OsAt10 gene increases expression of polyamines biosynthesis genes under cold stress. In transgenic A. thaliana plants, overexpression of the OsAt10 gene increased cold stress tolerance by regulating expression of stress marker genes, TBARS content, ion leakage level, antioxidant enzymes activity, and polyamines content, indicating that the OsAt10 gene could be economically important for improving low-temperature stress tolerance in plants.

     

Origin and evolution of genes related to ABA metabolism and its signaling pathways

Since plants cannot move to avoid stress, they have sophisticated acclimation mechanisms against a variety of abiotic stresses. The phytohormone abscisic acid (ABA) plays essential roles in abiotic stress tolerances in land plants. Therefore, it is interesting to address the evolutionary origins of ABA metabolism and its signaling pathways in land plants. Here, we focused on 48 ABA-related Arabidopsis thaliana genes with 11 protein functions, and generated 11 orthologous clusters of ABA-related genes from A. thaliana, Arabidopsis lyrata, Populus trichocarpa, Oryza sativa, Selaginella moellendorffii, and Physcomitrella patens. Phylogenetic analyses suggested that the common ancestor of these six species possessed most of the key protein functions of ABA-related genes. In two species (A. thaliana and O. sativa), duplicate genes related to ABA signaling pathways contribute to the expression variation in different organs or stress responses. In particular, there is significant expansion of gene families related to ABA in evolutionary periods associated with morphological divergence. Taken together, these results suggest that expansion of the gene families related to ABA signaling pathways may have contributed to the sophisticated stress tolerance mechanisms of higher land plants.