Divergent evolution of oxidosqualene cyclases in plants

Triterpenes are one of the largest classes of plant metabolites and have important functions. A diverse array of triterpenoid skeletons are synthesized via the isoprenoid pathway by enzymatic cyclization of 2,3-oxidosqualene. The genomes of the lower plants Chlamydomonas reinhardtii and moss (Physcomitrella patens) contain just one oxidosqualene cyclase (OSC) gene (for sterol biosynthesis), whereas the genomes of higher plants contain nine to 16 OSC genes. Here we carry out functional analysis of rice OSCs and rigorous phylogenetic analysis of 96 OSCs from higher plants, including Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium distachyon. The functional analysis identified an amino acid sequence for isoarborinol synthase (OsIAS) (encoded by Os11g35710/OsOSC11) in rice. Our phylogenetic analysis suggests that expansion of OSC members in higher plants has occurred mainly through tandem duplication followed by positive selection and diversifying evolution, and consolidated the previous suggestion that dicot triterpene synthases have been derived from an ancestral lanosterol synthase instead of directly from their cycloartenol synthases. The phylogenetic trees are consistent with the reaction mechanisms of the protosteryl and dammarenyl cations which parent a wide variety of triterpene skeletal types, allowing us to predict the functions of the uncharacterized OSCs.     

Use of genomic history to improve phylogeny and understanding of births and deaths in a gene family

Polyploidy events have played an important role in the evolution of angiosperm genomes. Here, we demonstrate how genomic histories can increase phylogenetic resolution in a gene family, specifically the expansin superfamily of cell wall proteins. There are 36 expansins in Arabidopsis and 58 in rice. Traditional sequence-based phylogenetic trees yield poor resolution below the family level. To improve upon these analyses, we searched for gene colinearity (microsynteny) between Arabidopsis and rice genomic segments containing expansin genes. Multiple rounds of genome duplication and extensive gene loss have obscured synteny. However, by simultaneously aligning groups of up to 10 potentially orthologous segments from the two species, we traced the history of 49 out of 63 expansin-containing segments back to the ancestor of monocots and eudicots. Our results indicate that this ancestor had 15-17 expansin genes, each ancestral to an extant clade. Some clades have strikingly different growth patterns in the rice and Arabidopsis lineages, with more than half of all rice expansins arising from two ancestral genes. Segmental duplications, most of them part of polyploidy events, account for 12 out of 21 new expansin genes in Arabidopsis and 16 out of 44 in rice. Tandem duplications explain most of the rest. We were also able to estimate a minimum of 28 gene deaths in the Arabidopsis lineage and nine in rice. This analysis greatly clarifies expansin evolution since the last common ancestor of monocots and eudicots and the method should be broadly applicable to many other gene families.     

Evolutionary relationship of plant catalase genes inferred from exon-intron structures: isozyme divergence after the separation of monocots and dicots

In order to understand the molecular evolution of catalase genes in higher plants, we compared the exon-intron structures of 12 genomic sequences from six plant species. It was assumed that the putative single primordial catalase gene had seven introns, because only those catalase genes having this structure are found in the monocotyledonae and dicotyledonae classes. After the evolutionary divergence of monocots from dicots, consecutive duplication of the primordial gene followed by the differential loss of introns occurred in each class to form three (or possibly four in dicots) diverse isozyme genes. In monocots, three ancestral isozyme genes were formed before the divergence of ancestral rice and maize. One of the rice genes, CatA, has an entirely new short intron which was not found in any other plant catalase gene examined. We have investigated the existence of the intron in the CatA homolog in other rice species by polymerase chain reaction (PCR) analysis. One major PCR product was found with the genomic DNAs from O. sativa (indica and japonica types), O. rufipogon and O. glaberrima. DNAs from several accessions of O. longistaminata showed variation in both the number and size of the DNA fragments amplified. PCR analyses and sequencing of the PCR products revealed that there are several CatA homologs having different sequences in some accessions of O. longistaminata. We have extended our study to other species in the Poaceae. The results suggest that the gain of the intron, most likely by insertion of a retroposon, took place in the ancestral genome of rice after its evolutionary divergence from other ancestral cereals such as barley, wheat and oat. Received: 20 November 1997 / Accepted: 5 January 1998     

High throughput screening of mutants of oat that are defective in triterpene synthesis

The triterpenes are a large and diverse group of plant natural products that have important functions in plant protection and food quality, and a range of pharmaceutical and other applications. Like sterols, they are synthesised from mevalonate via the isoprenoid pathway, the two pathways diverging after 2,3-oxidosqualene. During triterpene synthesis 2,3-oxidosqualene is cyclised to one of a number of potential products, the most common of these being the pentacyclic triterpene β-amyrin. Plants often produce complex mixtures of conjugated triterpene glycosides which may be derived from a single triterpene skeleton. The delineation, functional analysis and exploitation of triterpene pathways in plants therefore represent a substantial challenge. Here we have carried out high throughput screening to identify mutants of diploid oat (Avena strigosa) that are blocked in the early steps of triterpene synthesis. We also show that mutants that are affected in the first committed step in synthesis of β-amyrin-derived triterpenes, and so are unable to cyclise 2,3-oxidosqualene to β-amyrin (sad1 mutants), accumulate elevated levels of primary sterols. The major differences were in Δ-7-campesterol and Δ-7-avenasterol, which both increased several fold relative to wild-type levels. This is presumably due to accumulation of squalene and 2,3-oxidosqualene and consequent feedback into the sterol pathway, and is consistent with previous reports in which specific oxidosqualene cyclase inhibitors and elicitors of triterpene biosynthesis were shown to have inverse effects on the flux through the sterol and triterpene pathways.     

Expression of functional oat phytochrome A in transgenic rice.

To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants.     

环氧角鲨烯环化酶在三萜化合物生物合成中的进展

三萜类化合物是植物代谢产物中最具多样性的化合物之一,具有广泛的生理活性和重要的经济价值.环氧角鲨烯环化酶(oxidosqualene cyclases,OSCs)催化2,3-氧化鲨烯环化生成不同类型的甾醇和植物三萜化合物,对天然产物的结构多样性具有重要意义.然而,目前对于OSCs酶催化2,3-氧化鲨烯发生环化多样性的机...     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice 10 kD Prolamin

Using PCR technique, two prolamin genes from Oryza sativa var. indica (cv. Guanglu′ ai) and O. sativa var. japonica (cv. Zhonghua 8) were amplified and cloned. The prolamin gene contained 525 base pairs and encoded 134 amino acid residues. The two genes cloned from two different rice cultivars exhibited 100% homology and were highly homologous with the 10 kD prolamin gene in other rice species amountin an homology ranging from 96.6% to 100%. The deduced amino acid sequence shared 34.2% homology with that of maize 10 kD prolamin. As for dicots, only two types of storage protein shared some homology with rice 10 kD prolamin. One was from Brazil nut and the other from castor bean. Analysis on the signal peptide of rice 10 kD prolamin showed that it shared higher homology with that of storage proteins in some monocots such as maize, sorghum and oat. No similar sequence was found in dicots. The gene sequences of "Guangluai” and "Zhonghua 8” 10 kD prolamin would appear in EMBL data-base under the accession number L36604 and L36605 respectively.     

Genome-wide analysis of WOX gene family in rice, sorghum, maize, Arabidopsis and poplar

WUSCHEL-related homeobox(WOX)genes form a large gene family specifically expressed in plants.They are known to play important roles in regulating the development of plant tissues and organs by determining cell fate.Recent available whole genome sequences allow us to do more comprehensive phylogenetic analysis of the WOX genes in plants.In the present study,we identified 11 and 21 WOXs from sorghum(Sorghum bicolor)and maize(Zea mays),respectively.The 72 WOX genes from rice(Oryza sativa),sorghum,maize,Arabidopsis(Arabidopsis thaliana)and poplar(Populus trichocarpa)were grouped into three well supported clades with nine subgroups according to the amino acid sequences of their homodomains.Their phylogenetic relationship was also supported by the observation of the motifs outside the homodomain.We observed the variation of duplication events among the nine sub-groups between monocots and eudicots,for instance,more gene duplication events of WOXs within subgroup A for monocots,while,less for dicots in this subgroup.Furthermore,we observed the conserved intron/exon structural patterns of WOX genes in rice,sorghum and Arabidopsis.In addition,WUS(Wuschel)-box and EAR(the ERF-associated amphiphilic repression)-like motif were observed to be conserved among several WOX subgroups in these five plants.Comparative analysis of expression patterns of WOX genes in rice and Arabidopsis suggest that the WOX genes play conserved and various roles in plants.This work provides insights into the evolution of the WOX gene family and is useful for future research.     

Together we stand: genes cluster to coordinate regulation

Although most eukaryotic genomes lack operons, occasionally clusters of genes are discovered that are related in function. Now, a metabolic operon-like gene cluster has been described in Arabidopsis thaliana, that is needed for triterpene synthesis.     

Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.     

Isolation, characterization and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid

Recently, a number of disease-resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease-resistance genes encoded a nucleotide-binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R -genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R -genes to amplify genomic sequences from ryegrass ( Lolium sp.). Sixteen NBS-like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P-Loop, kinase-2, kinase-3a, GLPL) present in other known plant NBS-leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non-toll and interleukin-1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R -genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R -genes in grass species.     

New Aspects on Lanosterol 14α-Demethylase and Cytochrome P450 Evolution: Lanosterol/Cycloartenol Diversification and Lateral Transfer

Sterol 14-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups. CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms. We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily. Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches. Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs. Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch. A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability). This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis. The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D. discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data. Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes. Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily. Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin.     

Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng

Panax ginseng produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane type and oleanane type. Dammarane-type ginsenosides dominate over oleanane type not only in amount but also in structural varieties. However, their sapogenin structure is restricted to two aglycones, protopanaxadiol and protopanaxatriol. So far, the genes encoding oxidosqualene cyclase (OSC) responsible for formation of dammarane skeleton have not been cloned, although OSC yielding oleanane skeleton (β-amyrin synthase) has been successfully cloned from this plant. In this study, cDNA cloning of OSC producing dammmarane triterpene was attempted from hairy root cultures of P. ginseng by homology based PCR method. A new OSC gene (named as PNA) obtained was expressed in a lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. LC-MS and NMR analyses identified the accumulated product in the yeast transformant to be dammarenediol-II, demonstrating PNA to encode dammarenediol-II synthase.     

Characterization of two rice genes for nuclear-encoded chloroplast ribosomal protein L12 and phylogenetic analysis of the acquisition of transit peptides and gene duplication

 We have identified two genes coding for chloroplast ribosomal protein L12 encoded in the nuclear genome of rice (Oryza sativa). These genes were designated rpl12-1 and rpl12-2 (rpl12, ribosomal protein L12). Northern analysis with specific probes revealed that both genes are transcribed. The expression of each gene seems to have a different regulatory machinery. It is also suggested that the expression of rpl12-1 is controlled in an organ-specific manner. The deduced amino-acid sequences of the mature peptide parts are more conserved than those of the transit peptide parts in both monocotyledonous and dicotyledonous plants. A phylogenetic tree was constructed using the nucleotide sequences of the transit peptide region of the rpl12s of reported plant. The tree includes estimates of when the transit peptides were acquired, and when the genes were duplicated, in the course of evolution. According to our hypothesis, the nuclear-translocated chloroplast ribosomal protein L12 gene obtained its transit peptide after the divergence of monocots and dicots, then gene duplications occurred independently in monocots and dicots, and subsequently rice and rye branched apart. Received: 4 October 1997 / Accepted: 5 January 1998