Molecular evolution and functional relevance of the chalcone synthase genes of pea

We have isolated seven genomic chalcone synthase (CHS) genes and six classes of CHS cDNA from elicitor-treated pea tissues. Comparison of the nucleotide sequences of the coding regions revealed the existence of eight members of the CHS gene family in pea. These can essentially be divided into three groups (PSCHS1, 2 and 8; PSCHS3, 4 and 5; and PSCHS6 and 7) on the basis of nucleotide and/or amino acid sequence comparisons of the coding regions, introns and promoter regions. We previously reported that the accumulation of CHS mRNAs is induced by elicitor treatment. Accumulation of CHS mRNA was observed mainly in roots and very little was found in floral organs. To specifically detect expression of each CHS gene in various types of pea cells, S1 nuclease protection assays were performed. Interestingly, the classification of the eight members of the CHS gene family based on the sequence identity was found to reflect their expression patterns as determined by the S1 nuclease protection assay. The first group of CHS genes, PSCHS1, 2 and 8, was strongly induced not only by elicitor treatment and UV irradiation but is also constitutively expressed in root and flower tissues. The second group, PSCHS3, 4 and 5, was also strongly induced by elicitor treatment and UV irradiation but is constitutively expressed only in root. Expression of the third group, PSCHS6 and 7 was barely detectable in any of the organs tested and was not influenced by environmental stimuli such as elicitor or UV. Furthermore, sequence analysis of the promoter region of each member of the CHS gene family revealed that putative cis-regulatory elements, such as Box-I, Box-II and G-Box, were conserved only in PSCHS1, 2, 3, 4 and 5. From these results we propose that an ancestral CHS gene might have given rise to defense response-related (UV irradiation- and elicitor-responsive) and -unrelated (unresponsive) genes at an early stage of evolution, followed by divergence within these subclasses based upon the developmental program in pea. Received: 5 November 1996 / Accepted: 6 February 1997     

Phytochrome and UV signal transduction pathways

The phytochromes are the best studied plant photoreceptors, controlling a wide variety of responses at both whole plant and single cell levels. Three signal transduction pathways, dependent on cGMP and/or calcium, have been found to be utilized by phytochrome to control the expression of genes required for chloroplast development (e.g., CAB and FNR) and anthocyanin biosynthesis (e.g., CHS). In particular, cGMP is a second messenger positively regulating CHS gene expression whilst calcium and calmodulin act as negative regulators. In addition to phytochrome regulation of CHS we have begun to examine the signal transduction pathways utilized by UV photoreceptors. In contrast to phytochrome-mediated responses, results indicate a role for calcium and calmodulin as positive regulators of CHS gene expression in UV light.     

Genome-wide analysis of the chalcone synthase superfamily genes of <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon–intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Genetics of homology-dependent gene silencing in Arabidopsis; a role for methylation

Ninety-eight independent transformed (T₁) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T₂ generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology- dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self-fertilization of plants hemizygous for the B and C-inserts suggested that these CHS-silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C-insert homozygotes was reduced to undetectable levels. Outcrosses of B- and C-insert homozygotes to wild-type plants resulted in F₁ plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild-type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F₁ plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C-insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild-type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C-insert-induced silencing and retained methylation and partial silencing after the silencing T-DNA was lost.     

Sexually selected UV signals in the tropical ornate jumping spider,Cosmophasis umbratica may incur costs from predation

Sexually selected ornaments and signals are costly to maintain if they are maladaptive in nonreproductive contexts. The jumping spider Cosmophasis umbratica exhibits distinct sexual dichromatism with males displaying elaborate UV body markings that signal male quality. Female C. umbratica respond favorably to UV‐reflecting males and ignore males that have their UV masked. However, Portia labiata, a UV‐sensitive spider‐eating specialist and a natural predator of C. umbratica, is known to use UV reflectance as a cue when hunting prey. We investigated the cost of these UV signals in C. umbratica in terms of their predation risk. Under experimental conditions, three choice scenarios were presented to P. labiata individuals. Choices by P. labiata were made between male C. umbratica with and without the UV signal; a UV‐reflecting male and non‐UV‐reflecting female; and a UV‐masked male and female. The presence and absence of UV signals was manipulated using an optical filter. Portia labiata exhibited a strong bias toward UV+ individuals. These results suggest the sexually selected trait of UV reflectance increases the visibility of males to UV‐sensitive predators. The extent of this male‐specific UV signal then is potentially moderated by predation pressure. Interestingly though, P. labiata still preferred males to females irrespective of whether UV reflectance was present or not. This suggests P. labiata can switch cues when conditions to detect UV reflectance are not optimal.     

Organization of the genes encoding chalcone synthase in Pisum sativum

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.     

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of -d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)₇CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.     

Characterization of a chalcone synthase (CHS) flower-specific promoter from Lilium orential ��Sorbonne��

The first enzyme in the flavonoid pathway, chalcone synthase, is encoded by a gene (CHS) whose expression is normally under developmental control. In our previous studies, an 896-bp promoter region of a flower-specific CHS gene was isolated from Lilium orential ‘Sorbonne’, and designated as PLoCHS. Here, the PLoCHS promoter was fused to the β-glucuronidase (GUS) gene to characterize its spatial and temporal expression in Petunia hybrida ‘Dreams Midnight’ using an Agrobacterium-mediated leaf disc transformation method. Our results demonstrated that GUS expression was present in flowers, but reduced or absent in the other tissues (leaf and stem) examined. In petals, GUS activity reached its peak at flower developmental stage 4, and decreased at later stages. Deletion analysis indicated that even a 307-bp fragment of the PLoCHS promoter could still direct flower-specific expression. Further deletion of the region from −261 to −72 bp resulted in weak expression in different organs, including flowers, leaves and stems. This evidence combined with prediction of cis-acting elements in the PLoCHS promoter suggests that the TACPyAT box located in this promoter plays a key role in the regulation of organ-specific expression.     

fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome-regulated genes in fhy1. In far-red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A-deficient mutant, confirming that gene expression in far-red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light-induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A-mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue-light signaling pathway for CHS expression.     

Genomic transcriptional response to loss of chromosomal supercoiling in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.