Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.     

Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum)

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T₁ plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.     

Isolation of a Suppressor-mutator/Enhancer-like transposable element, Tpn1, from Japanese morning glory bearing variegated flowers.

The Japanese morning glory has an extensive history of genetic studies. Many mutants in the colors and shapes of its flowers and leaves have been isolated since the 17th century, and more than 200 genetic loci have been localized for the 10 linkage groups. They include over 20 mutable loci, several with variegated flower phenotypes. In a line of Japanese morning glory bearing variegated flowers called flecked, a transposable element of 6.4 kb, termed Tpn1, was found within one of the anthocyanin biosynthesis genes encoding dihydroflavonol-4-reductase (DFR). The 6.4-kb element carries 28-bp perfect terminal inverted repeats, the outer 13 bp being identical to those of the maize transposable element Suppressor-mutator/Enhancer. It is flanked by 3-bp direct repeats within the second intron of the DFR gene, 9 bp upstream of the third exon. When somatic and germinal excision occurs, it produces excision sequences characteristic of plant transposable elements. Cosegregation data of the variegated flower phenotype and the DFR gene carrying Tpn1 indicated that the mutable phenotype is due to excision of Tpn1 from the DFR gene. Sequences homologous to Tpn1 are present in multiple copies in the genome of Japanese morning glory.     

Developmental changes of sepiapterin synthase activity associated with a variegated purple gene in Drosophila melanogaster

A variegated position effect on the autonomous gene, purple, has been studied enzymologically in Drosophila melanogaster. Sepiapterin synthase, the enzyme system associated with pr ⁺, was examined for activity in different developmental stages of the fly. The results indicate that T(Y:2) pr ^c5, cn/pr^c4 cn flies (flies in which pr ⁺ has been translocated and which exhibit variegation) have a reduced amount of enzyme activity as compared with both Oregon-R and pr ¹ flies. This reduction in activity was not found in larval stages, which suggests that the inactivation process probably occurs in late larval or early pupal stages. The phenotype of the variegated adult has white eyes with red-colored spots and patches where drosopterins occur. The phenotype of the fly carrying the translocation is modified by the presence of additional Y chromosomes. This extends the observation from other systems that extra heterochromatin acts to suppress the variegated position effect. The advantages of studying the variegation by measuring enzyme activity, as well as the phenotypic expression, are several; for example, the developmental time at which variegation occurs may be estimated even though drosopterin synthesis is not occurring.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the Department of Energy under Contract No. W-7405-eng-26.     

A candidate male-fertility female-fertility gene tagged by the soybean endogenous transposon, Tgm9

In soybean, the W4 gene encoding dihydroflavonol-4-reductase controls anthocyanin pigment biosynthesis in flowers. The mutant allele, w4-m, is characterized by variegated flowers and was evolved from the insertion of an endogenous transposable element, Tgm9, in intron II of the W4 gene. In the w4-m mutant line, reversion of the unstable allele from variegated to normal purple flower in revertants would indicate Tgm9’s excision accompanied by its insertion into a second locus. We identified a male-sterile, female-sterile mutant from such germinal revertant bearing purple flowers. The objectives of our investigation were to map the sterility locus, identify candidate genes for the male-fertile, female-fertile phenotype, and then determine if sterility is associated with the insertion of Tgm9 in the sterility locus. We used bulked segregant analysis to map the locus to molecular linkage group J (chromosome 16). Fine mapping enabled us to flank the locus to a 62-kb region that contains only five predicted genes. One of the genes in that region, Glyma16g07850.1, codes for a helicase. A rice homolog of this gene has been shown to control crossing over and fertility phenotype. Thus, Glyma16g07850.1 is most likely the gene regulating the male and female fertility phenotype in soybean. DNA blot analysis of the segregating individuals for Tgm9 showed perfect association between sterility and the presence of the transposon. Most likely, the sterility mutation was caused by the insertion of Tgm9. The transposable element should facilitate identification of the male- and female-fertility gene. Characterization of the fertility gene will provide vital molecular insight on the reproductive biology of soybean and other plants.     

Nuclear Gene-Induced Plastome Mutations in OENOTHERA HOOKERI. I. Genetic Analysis

A nuclear gene mutation in Oenothera hookeri increases the frequency of variegated sectors. The gene is recessive; the variegation is cytoplasmically transmitted. Once variegation is induced, the mutant gene is not required for its continued expression. The induced sectors may differ one from another. The gene expresses unique patterns of penetrance and of maternal effect. The genetic data implicate the chloroplasts as the site for the expression of variegation. The chloroplasts of O. parviflora are also subject to the action of the nuclear gene. Possible mechanisms by which a gene might cause chloroplasts to mutate are discussed.     

Somatic Variegation and Germinal Mutability Reflect the Position of Transposable Element Dissociation within the Maize R Gene

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.     

Both Positive and Negative Selection Pressures Contribute to the Polymorphism Pattern of the Duplicated Human CYP21A2 Gene

The human steroid 21-hydroxylase gene (CYP21A2) participates in cortisol and aldosterone biosynthesis, and resides together with its paralogous (duplicated) pseudogene in a multiallelic copy number variation (CNV), called RCCX CNV. Concerted evolution caused by non-allelic gene conversion has been described in great ape CYP21 genes, and the same conversion activity is responsible for a serious genetic disorder of CYP21A2, congenital adrenal hyperplasia (CAH). In the current study, 33 CYP21A2 haplotype variants encoding 6 protein variants were determined from a European population. CYP21A2 was shown to be one of the most diverse human genes (HHe=0.949), but the diversity of intron 2 was greater still. Contrary to previous findings, the evolution of intron 2 did not follow concerted evolution, although the remaining part of the gene did. Fixed sites (different fixed alleles of sites in human CYP21 paralogues) significantly accumulated in intron 2, indicating that the excess of fixed sites was connected to the lack of effective non-allelic conversion and concerted evolution. Furthermore, positive selection was presumably focused on intron 2, and possibly associated with the previous genetic features. However, the positive selection detected by several neutrality tests was discerned along the whole gene. In addition, the clear signature of negative selection was observed in the coding sequence. The maintenance of the CYP21 enzyme function is critical, and could lead to negative selection, whereas the presumed gene regulation altering steroid hormone levels via intron 2 might help fast adaptation, which broadly characterizes the genes of human CNVs responding to the environment.     

Natural foliar variegation without costs? The case of Begonia

Background and Aims

Foliar variegation is recognized as arising from two major mechanisms: leaf structure and pigment-related variegation. Begonia has species with a variety of natural foliar variegation patterns, providing diverse examples of this phenomenon. The aims of this work are to elucidate the mechanisms underlying different foliar variegation patterns in Begonia and to determine their physiological consequences.

Methods

Six species and one cultivar of Begonia were investigated. Light and electron microscopy revealed the leaf structure and ultrastructure of chloroplasts in green and light areas of variegated leaves. Maximum quantum yields of photosystem II were measured by chlorophyll fluorescence. Comparison with a cultivar of Ficus revealed key features distinguishing variegation mechanisms.

Key Results

Intercellular space above the chlorenchyma is the mechanism of variegation in these Begonia. This intercellular space can be located (a) below the adaxial epidermis or (b) below the adaxial water storage tissue (the first report for any taxa), creating light areas on a leaf. In addition, chlorenchyma cell shape and chloroplast distribution within chlorenchyma cells differ between light and green areas. Chloroplasts from both areas showed dense stacking of grana and stroma thylakoid membranes. The maximum quantum yield did not differ significantly between these areas, suggesting minimal loss of function with variegation. However, the absence of chloroplasts in light areas of leaves in the Ficus cultivar led to an extremely low quantum yield.

Conclusions

Variegation in these Begonia is structural, where light areas are created by internal reflection between air spaces and cells in a leaf. Two forms of air space structural variegation occur, distinguished by the location of the air spaces. Both forms may have a common origin in development where dermal tissue becomes loosely connected to mesophyll. Photosynthetic functioning is retained in light areas, and these areas do not include primary veins, potentially limiting the costs of variegation.     

CYP46 T/C Polymorphism is not Associated with Alzheimer’s Dementia in a Population from Hungary

Multiple genetic and environmental factors regulate the susceptibility to Alzheimer’s disease (AD). Recently, several independent studies have reported that a locus on chromosome 14q32.1, where a gene encoding a cholesterol degrading enzyme of the brain, called 24-hydroxylase (CYP46A1) is located, has been linked with AD. The single nucleotide polymorphism (T/C) in intron 2 of CYP46 gene has been found to confer the risk for AD. The water soluble 24(S)-hydroxysterol is the product of the CYP46A1, and elevated plasma and cerebrospinal fluid hydroxysterol concentrations have been found in AD, reflecting increased brain cholesterol turnover or cellular degradation, due to the neurodegenerative process. A case–control study was performed on 125 AD and 102 age- and gender-matched control subjects (CNT) from Hungary, to test the association of CYP46 T/C and apolipoprotein E (ApoE) gene polymorphisms in AD. The frequency of the CYP46 C allele was similar (χ²=0.647, df=1, P=0.421, exact P=0.466, OR=0.845; 95% CI: 0.561–1.274) in both groups (CNT: 27%; 95% CI: 21.3–33.4; AD 30%; 95% CI: 25.0–36.3). The ApoE ɛ4 allele was significantly over-represented (χ²=11.029, df=2, P=0.004) in the AD population (23.2%; 95% CI: 18.2–29.0) when compared with the CNT (11.3%; 95% CI: 7.4–16.6). The presence or absence of one or two CYP46C alleles together with the ApoE ɛ4 allele did not increase the risk of AD (OR=3.492; 95% CI: 1.401–8.707; P<0.007 and OR=3.714; 95% CI: 1.549–8.908; P<0.003, respectively). Our results indicate that the intron 2 T/C polymorphism of CYP46 gene (neither alone, nor together with the ɛ4 allele) does not increase the susceptibility to late-onset sporadic AD in the Hungarian population.     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5′ end of the element, and 33 copies of the sequence motif lie within 800 by of the 3′ terminus. All these 22 copies of the sequence motif near the 5′ terminus and 30 copies in the 3′ terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5′ and 3′ subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

三色(Bougainvillea peruviana‘Thimma’)在转录水平的甜菜色素和类黄酮积累比较

Bougainvillea peruviana‘Thimma’属于三角梅属,该属植物积累甜菜色素而不是像绝大多数高等植物一样积累花青素。该材料特征同株出现3种颜色：白色、洋红色和白/洋红相间。本研究首次使用3种花色特征的花序（红色Yp、混合色的Ym、白色Yw）作为研究材料进行高通量测序。并通过real-time PCR方法对探测到的花色代谢基因进行验证。共获得平均长度为616 bp的73 325条基因。3种材料的差异显示基因（DEGs）中有327个被注释到甜菜色素合成基因,308个被注释到类黄酮合成基因,466个被注释到花青素合成基因。我们选出8个基因：4个甜菜色素合成基因（PPO,CYP76AD1,cDOPA-5-GT,DODA）和4个花青素合成基因（FLS,DFR,LDOX,3-GT）进行验证。其中,4个甜菜色素合成基因在3种花色材料中的表达较好的正相关于甜菜色素含量。花青素合成途径末端的3个基因（DFR,LDOX,3-GT）在B.peruviana中首次被验证。real-time PCR的验证结果很好的吻合转录组测序的结果。同时,B.peruviana也提供了一个很好的三角梅属植物的生理、生化和分子生物学研究的工具,有效的摒除其他生物学干扰。     

Antioxidative response of Golden Agave leaves with different degrees of variegation under high light exposure

Variegated mutants are spotted rare in wild, and especially in the high light (HL) conditions. In addition, these plants are cultivated by a growing number of horticulturists due to their high economic value. In this experiment, the antioxidative response of both albino and green tissues from a mutant plant Golden Agave, which possesses leaves with different degrees of variegation, was investigated under normal or HL exposure, respectively. Severe oxidative stress was observed in green tissues of high albinism leaves (30?C40?% albino tissues, muy) over low albinism counterparts (less than 10?% albino tissues, mug). Compared with green tissues, low oxidative damage (relative electrolyte leakage and protein damage) and antioxidant enzyme (SOD, CAT, APX, and GR) activities were observed in the albino than in the adjacent green tissues. Photoinactivation of the CAT and the APX enzyme activities were observed in the albino tissues to a significantly level under HL exposure. In addition, lower redox values (ASC/DHA and GSH/GSSG) and higher levels of reactive oxygen species (ROS) were monitored in the green tissues of the muy over mug mutant even under normal light conditions. This work could help us to understand the HL sensitivity of variegated mutant plants better and allow us to improve variegated mutant plant cultivation skills. At last, the ??oxidative stress hypothesis?? was introduced to illustrate the reduced evolutionary advantage of plant variegation phenomenon in the discussion.     

An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3′-hydroxylase gene

The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3′H, the gene for flavonoid 3′-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.