Contrasting effects of selection on sequence diversity and linkage disequilibrium at two phytoene synthase loci

We investigated the effects of human selection for yellow endosperm color, representing increased carotenoid content, on two maize genes, the Y1 phytoene synthase and PSY2, a putative second phytoene synthase. Multiple polymorphic sites were identified at Y1 and PSY2 in 75 white and yellow maize inbred lines. Many polymorphic sites showed strong association with the endosperm color phenotype at Y1, but no detectable association was found at PSY2. Nucleotide diversity was equivalent for whites and yellows at PSY2 but was 19-fold less in yellows than in whites at Y1, consistent with the white ancestral state of the gene. The strong sequence haplotype conservation within yellows at Y1 and a significant, negative Tajima's D both verified positive selection for yellow endosperm. We propose that two independent gain-of-function events associated with insertions into the promoter of the Y1 gene and upregulation of expression in endosperm have been incorporated into yellow maize.     

Intragenic Enhancers and Suppressors of Phytoene Desaturase Mutations in Chlamydomonas reinhardtii

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis-the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.     

Cryptochrome 1 contributes to blue-light sensing in pea

Cryptochromes are widespread in higher plants but their physiological roles as blue-light photoreceptors have been examined in relatively few species. Screening in a phyA null mutant background has identified several blue-light response mutants in pea (Pisum sativum), including one that carries a substitution of a highly conserved glycine residue in the N-terminal photolyase-homologous domain of the pea CRY1 gene. Analyses of cry1, phyA, and phyB mutants show that all three photoreceptors contribute to seedling photomorphogenesis under high-irradiance blue light, whereas phyA is the main photoreceptor active under low irradiances. Triple phyA phyB cry1 mutants grown under high-irradiance blue light are indistinguishable from dark-grown wild-type plants in length and leaf expansion but show a small residual response to higher-irradiance white light. Monogenic cry1 mutants have little discernable phenotype at the seedling stage, but later in development are more elongated than wild-type plants. In addition, the loss of cry1 moderates the short-internode phenotype of older phyA mutants, suggesting an antagonism between phyA and cry1 under some conditions. Pea cry1 has a small inhibitory effect on flowering under long and short days. However, the phyA cry1 double mutant retains a clear promotion of flowering in response to blue-light photoperiod extensions, indicating a role for one or more additional blue-light photoreceptors in the control of flowering in pea.     

The gene crtI mediates the conversion of phytoene into colored carotenoids in Rhodopseudomonas capsulata

Carotenoids are membrane pigments present in all photosynthetic organisms, providing essential photoprotective functions. The first carotenoid formed in the pathway is phytoene, a colorless compound which is then converted into colored carotenoids by a series of dehydrogenation reactions. In the photosynthetic bacterium Rhodopseudomonas capsulata mutations that affect carotenoid biosynthesis before colored carotenoids are formed have a "blue-green" phenotype as opposed to the "red" of wild type cells. We have extracted carotenoids from several blue-green mutants and found that two strains (BPY69 and BPY102) accumulate phytoene and no colored carotenoids. These mutants failed to dehydrogenate phytoene in an in vitro assay. However, dehydrogenation of this compound can be achieved in vitro by adding a cell-free extract from another blue-green mutant blocked earlier in the pathway. Genetic complementation and deletion mapping indicate that the gene crtI is responsible for the conversion of phytoene into colored carotenoids in these mutants.     

A "White" Anthocyanin-less Pomegranate (Punica granatum L.) Caused by an Insertion in the Coding Region of the Leucoanthocyanidin Dioxygenase (LDOX; ANS) Gene

Color is an important determinant of pomegranate fruit quality and commercial value. To understand the genetic factors controlling color in pomegranate, chemical, molecular and genetic characterization of a "white" pomegranate was performed. This unique accession is lacking the typical pomegranate color rendered by anthocyanins in all tissues of the plant, including flowers, fruit (skin and arils) and leaves. Steady-state gene-expression analysis indicated that none of the analyzed "white" pomegranate tissues are able to synthesize mRNA corresponding to the PgLDOX gene (leucoanthocyanidin dioxygenase, also called ANS, anthocyanidin synthase), which is one of the central structural genes in the anthocyanin-biosynthesis pathway. HPLC analysis revealed that none of the "white" pomegranate tissues accumulate anthocyanins, whereas other flavonoids, corresponding to biochemical reactions upstream of LDOX, were present. Molecular analysis of the "white" pomegranate revealed the presence of an insertion and an SNP within the coding region of PgLDOX. It was found that the SNP does not change amino acid sequence and is not fully linked with the "white" phenotype in all pomegranate accessions from the collection. On the other hand, genotyping of pomegranate accessions from the collection and segregating populations for the "white" phenotype demonstrated its complete linkage with the insertion, inherited as a recessive single-gene trait. Taken together, the results indicate that the insertion in PgLDOX is responsible for the "white" anthocyanin-less phenotype. These data provide the first direct molecular, genetic and chemical evidence for the effect of a natural modification in the LDOX gene on color accumulation in a fruit-bearing woody perennial deciduous tree. This modification can be further utilized to elucidate the physiological role of anthocyanins in protecting the tree organs from harmful environmental conditions, such as temperature and UV radiation.     

Control of chloroplast redox by the IMMUTANS terminal oxidase

Variegation mutants offer excellent opportunities to study interactions between the nucleus-cytoplasm, the chloroplast, and the mitochondrion. Variegation in the immutans ( im ) mutant of Arabidopsis is induced by a nuclear recessive gene and the extent of variegation can be modulated by light and temperature. Whereas the green sectors have morphologically normal chloroplasts, the white sectors are devoid of pigments and accumulate a colourless carotenoid, phytoene. The green sectors are hypothesized to arise from cells that have avoided irreversible photooxidative damage whereas the white sectors originate from cells that are photooxidized. Cloning of the IMMUTANS ( IM ) gene has revealed that IMMUTANS (IM) is a plastid homologue of the mitochondrial alternative oxidase. This finding suggested a model in which IM functions as a redox component of the phytoene desaturation pathway, which requires phytoene desaturase activity. Consistent with this idea, IM has quinol oxidase activity in vitro. Recent studies have revealed that IM plays a more global role in plastid metabolism. For example, it appears to be the elusive terminal oxidase of chlororespiration and also functions as a light stress protein.     

Characterization of a zebra mutant of rice with increased susceptibility to light stress

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.     

Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation.

Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.     

Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection

The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5' end and a chitin synthase (class V)-encoding region at its 3' end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5' upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25 degrees C but have severe growth and cellular abnormalities at 37 degrees C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Delta mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Delta mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37 degrees C and is of critical importance to its virulence.     

A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.     

Different Roles for Phytochrome in Etiolated and Green Plants Deduced from Characterization of Arabidopsis thaliana Mutants

We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A. thaliana hy mutants. Among six complementation groups that define the HY phenotype in A. thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants. When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed. Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A. thaliana seedlings. When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome. An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally. Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (e.g., gene expression) associated with chloroplast development.     

甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达

八氢番茄红素脱氢酶( CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用.以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸.与来自M.populi BJ001、M.chloromethanicum CM4和M.extorquens AM1的crtI一致性均为93％.将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI.测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40％.实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考.