Expression In Petals Research Articles

The two non-functional myrosinase genes TGG3 and TGG6 in Arabidopsis are expressed predominantly in pollen

Myrosinases are a family of β-thioglucoside glucohydrolases (TGGs) that catalyze hydrolysis of glucosinolates to deter pest insects and pathogens. Arabidopsis has six myrosinase genes, of which TGG1, TGG2, TGG4 and TGG5 have been reported as functional, and TGG3 as a pseudogene, while TGG6 has received little attention. Both genomic and cDNA of TGG6 were cloned from five ecotypes and sequenced in this research. TGG6 had the same gene structure as TGG4 and TGG5, but contained several frameshift mutations and should be non-functional. Reverse transcription polymerase chain reaction indicated that TGG6 was specifically expressed in flowers. Promoter-driven GUS expression showed that TGG6 was solely expressed in mature pollen grains when flowers initiated blossom. TGG6 expression was not detected in other cell types of anthers and other organs. The TGG3 promoter-driven GUS expression was similarly predominantly expressed in mature pollen, but the expression of TGG3 in pollen was initiated before flower bud blossoming, earlier than of TGG6, and there was weak expression in petals and pollen sac cells for a short period.

“Yellow Jimba”: Suppression of Carotenoid Cleavage Dioxygenase (CmCCD4a) Expression Turns White Chrysanthemum Petals Yellow

‘Jimba’ is the most popular white-flowered chrysanthemum cultivar in Japan. A yellow-flowered cultivar with the same growth properties as ‘Jimba’ will benefit growers because both forms could be produced under the same conditions. Many breeders have therefore tried to produce a “Yellow Jimba” by mutation breeding but have not yet succeeded. Previously, we showed that color mutation from white to yellow in the petals of ray florets is caused by the loss of a carotenoid cleavage dioxygenase gene, CmCCD4a. Here we introduced two separate CmCCD4a RNAi constructs into ‘Jimba’ by Agrobacterium-mediated transformation. The double-transformation effectively suppressed CmCCD4a expression in petals, which became yellow. The yellowest transformants contained 102 μg·g−1 FW carotenoids in the petals, and the expression level of CmCCD4a was 0.4% of the wild-type level. Although the transformed plants were significantly smaller than the wild type, flower size was unchanged. We consider that CmCCD4a could become a powerful tool for petal color manipulation from white to yellow.

Characterization and functional analysis of a novel PCP gene BcMF5 from Chinese cabbage ( Brassica campestris L. ssp. chinensis Makino)

Brassica campestris Male Fertile 5 (BcMF5), a novel member of the pollen coat protein class A (PCP-A) gene family, was identified from Brassica campestris L. ssp. chinensis Makino (Chinese cabbage-pak-choi). Temporal and spatial expression analysis showed that BcMF5 is a late-expressed PCP gene related to the process of determining pollen fertility. Functional analysis by hairpin RNA (hpRNA)-mediated RNA interference also showed that the expression of BcMF5 is inhibited, which resulted in the low germination ability of the pollen and also in an abnormality of the pollen exemplified by a collapsed germination furrow. This demonstrates that the expression of BcMF5 is closely related to the tapetum. Further, the expression profile of the BcMF5 promoter in Arabidopsis was also analyzed. This analysis indicated that the BcMF5 promoter began expression in the early stage of anther development and drove high levels of glucuronidase (GUS) expression in anthers, pollen, and the pollen tube in the late stage of pollen development, but did not drive any expression in petals, sepals, or pistils. Together with the functional analysis, the hypothesis that BcMF5 may have a sporophytic or gametophytic expression pattern is presented.

Sucrose prevents up-regulation of senescence-associated genes in carnation petals

cDNA microarrays were used to characterize senescence-associated gene expression in petals of cut carnation (Dianthus caryophyllus) flowers, sampled from anthesis to the first senescence symptoms. The population of PCR fragments spotted on these microarrays was enriched for flower-specific and senescence-specific genes, using subtractive hybridization. About 90% of the transcripts showed a large increase in quantity, approximately 25% transiently, and about 65% throughout the 7 d experiment. Treatment with silver thiosulphate (STS), which blocks the ethylene receptor and prevented the normal senescence symptoms, prevented the up-regulation of almost all of these genes. Sucrose treatment also considerably delayed visible senescence. Its effect on gene expression was very similar to that of STS, suggesting that soluble sugars act as a repressor of ethylene signal transduction. Two fragments that encoded a carnation EIN3-like (EIL) protein were isolated, some of which are key transcription factors that control ethylene response genes. One of these (Dc-EIL3) was up-regulated during senescence. Its up-regulation was delayed by STS and prevented by sucrose. Sucrose, therefore, seems to repress ethylene signalling, in part, by preventing up-regulation of Dc-EIL3. Some other transcription factors displayed an early increase in transcript abundance: a MYB-like DNA binding protein, a MYC protein, a MADS-box factor, and a zinc finger protein. Genes suggesting a role in senescence of hormones other than ethylene encoded an Aux/IAA protein, which regulate transcription of auxin-induced genes, and a cytokinin oxidase/dehydrogenase, which degrades cytokinin. Taken together, the results suggest a master switch during senescence, controlling the co-ordinated up-regulation of numerous ethylene response genes. Dc-EIL3 might be (part of) this master switch.

Convergence, constraint and the role of gene expression during adaptive radiation: floral anthocyanins in Aquilegia

Convergent phenotypes are testament to the role of natural selection in evolution. However, little is known about whether convergence in phenotype extends to convergence at the molecular level. We use the independent losses of floral anthocyanins in columbines (Aquilegia) to determine the degree of molecular convergence in gene expression across the anthocyanin biosynthetic pathway (ABP). Using a phylogeny of the North American Aquilegia clade, we inferred six independent losses of floral anthocyanins. Via semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we monitored developmental and tissue-specific variation in expression of the six major structural ABP loci in three Aquilegia species, two that produce anthocyanins (A+) and one that does not (A-). We then compared ABP expression in petals of old-bud and pre-anthesis flowers of 13 Aquilegia species, eight wild species and two horticultural lines representing seven independent A- lineages as well as three wild A+ species. We only found evidence of down-regulation of ABP loci in A- lineages and losses of expression were significantly more prevalent for genes late in the pathway. Independent contrast analysis indicates that changes in expression of dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) are strongly phylogenetically correlated consistent with the multilocus targets of trans-regulatory elements in the ABP of other systems. Our findings strongly suggest that pleiotropy constrains the evolution of loss of floral anthocyanins to mutations affecting genes late in the ABP mostly through convergent changes in regulatory genes. These patterns support the hypothesis that rapid evolutionary change occurs largely through regulatory rather than structural mutations.

A homolog of the defender against apoptotic death gene ( DAD1) in senescing gladiolus petals is down-regulated prior to the onset of programmed cell death

We isolated a homolog of the potential anti-apoptotic gene, defender against apoptotic death (DAD1) from gladiolus petals as full-length cDNA (GlDAD1), and investigated the relationship between its expression and the execution processes of programmed cell death (PCD) in senescing petals. RNA gel blotting showed that GlDAD1 expression in petals was drastically reduced, considerably before the first visible senescence symptom (petal wilting). A few days after down-regulation GlDAD1 expression, DNA and nuclear fragmentation were observed, both specific for the execution phase of PCD.

Ethylene biosynthetic genes are differentially regulated by ethylene and ACC in carnation styles

Treatment of intact carnation (Dianthus caryophyllus‘White Sim’) flowers with ethylene induced increased ethylene production from all flower organs and increased accumulation of transcripts from genes encoding enzymes involved in ethylene biosynthesis. Three members of the 1-aminocyclopropane-1-carboxylate (ACC) synthase gene family (DCACS1, 2 and 3) were differentially regulated by ethylene within the flower. Treatment of isolated flower organs with 10 µL L−1 ethylene for 24 h, enhanced ethylene production in ovaries and petals, but not styles. Transcripts from DCACS2, DCACS3, and DCACO1 (ACC oxidase 1) were increased in isolated treated styles, although not to the levels detected in styles from ethylene-treated flowers. Although ethylene treatment of isolated styles did not induce elevated ethylene biosynthesis, treatment with 100 µM ACC resulted in large increases in ethylene evolution from the style. ACC treatment induced increases in mRNA levels of DCACS1, DCACS2, DCACS3 and DCACO1. When flowers were pretreated with CoCl2, to prevent the conversion of ACC to ethylene, DCACS1, DCACS2, and DCACO1 transcripts were still induced, although to a lesser degree than following ACC treatment alone. Similar results were seen when flowers were treated with the ethylene action inhibitor, 1-methylcyclopropene (1-MCP), prior to ACC treatment. In styles, DCACS2, DCACS3, and DCACO1 genes were transcriptionally regulated by both ACC and ethylene. In petals, ACC increased transcript abundance of all 4 genes, but these transcripts were not detectable when flowers were pretreated with either CoCl2 or 1-MCP. These results confirm that ethylene is the primary regulator of ACS and ACO gene expression in petals.

Cell-type-specific expression of plant cytochrome c mRNA in developing flowers and roots.

We have used RNA in situ hybridization to analyze the expression of transcripts encoding cytochrome c in different tissues and organs of sunflower (Helianthus annuus). Although northern-blot hybridization experiments indicate that the relative abundance of transcripts does not vary greatly, we have detected important changes in localization during flower development. Enhanced expression is observed in floral meristems as soon as they are discernible from the central portion of the capitulum containing the inflorescence meristem. As flowers develop, labeling is observed in all developing floral organ primordia. Later in development, expression in petals is reduced, and only the central portion of the flower becomes labeled. During the process of stamen formation, hybridization signals were obtained mainly in anthers. Less developed flowers at this stage showed expression through the archesporial tissue. During meiosis, the label was observed mainly in tapetal cells. Specific expression patterns, similar to those obtained for sunflower, were observed when Arabidopsis flowers were analyzed with a homologous cytochrome c probe. Specific patterns of expression were also observed in young sunflower roots. In this case, enhanced expression was detected in developing endodermis and pericycle and in protoxylem initials. We conclude that cell-specific mechanisms operate to regulate the abundance of cytochrome c encoding transcripts in different plant tissues. The overlap between the expression patterns of the nuclear encoded cytochrome c gene and some mitochondrial genes suggests the existence of coordinated mechanisms of expression.

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.

Isolation and characterisation of the carnation floral-specific MADS box gene, CMB2

The cDNA clone KD81, was isolated from a carnation petal cDNA library based on its strong differential expression in petals compared with leaves. The deduced amino acid sequence of KD81 indicated high homology with members of the MADS box family of transcription factors. Identified within the deduced amino acid sequence are two conserved domains; an N-terminal, MADS box and a central, K box. The gene encoding KD81 was termed Carnation MADS Box gene 2 ( CMB2). Expression of CMB2 is floral-specific and in petal, transcripts were persistent from the initial stages of development through flower opening. Transcripts were not detected in vegetative tissues. The CMB2 protein is most homologous to TDR6 from tomato, the product of the petal and stamen identity gene DEFICIENS ( DEFA), and several DEFA homologues including SLM3, STDEF, PMADS1 and APETALA3. Southern blot analysis indicated that CMB2 is present as a single copy within the carnation genome. Characterisation of a genomic clone encoding CMB2, revealed the molecular structure of CMB2 to be consistent with that reported for other plant MADS box genes. Analysis of the CMB2 promoter sequence revealed the presence of two putative cis-acting elements known as serum response elements (SREs). These elements are proposed as the target for MADS box domain binding and may be involved in the regulation/autoregulation of gene expression. CMB2 represents the first reported isolation of a MADS box gene from carnation.

The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects.

APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box. The APETALA3 promoter contains three close matches to the consensus CArG box sequence. To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions. 496 base pairs of APETALA3 promoter sequence 5' to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers. A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to petals and stamens in the flower. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects. Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors. Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged. By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor.

A MADS box transcription factor of the AP1/AGL9 subfamily is also expressed in the seed coat of pea (Pisum sativum) during development.

As part of a programme to identify seed coat-specific transcripts in pea (Pisum sativum cv. Finale) using the differential display method, we identified and isolated a cDNA encoding a MADS box transcription factor. The predicted peptide contains 247 amino acids with the conserved N-terminal MADS box and is closely related to the AP1/AGL9 subfamily of the MADS box proteins. In addition to weak expression in petals, stamens and carpels, this MADS box mRNA is specifically expressed in the seed coat during seed development.

Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence

The senescence of carnation (Dianthus caryophyllus L.) flower petals is regulated by the phytohormone ethylene and is associated with considerable catabolic activity including the loss of protein. In this paper we present the molecular cloning of a cysteine proteinase and show that its expression is regulated by ethylene and associated with petal senescence. A 1600 bp cDNA was amplified by polymerase chain reaction using a 5'-specific primer and 3'-nonspecific primer designed to amplify a 1-aminocyclopropane-1-carboxylate synthase cDNA from reverse-transcribed stylar RNA. The nucleotide sequence of the cloned product (pDCCP1) was found to share significant homology to several cysteine proteinases rather than ACC synthase. A single open reading frame of 428 amino acids was shown to share significant homology with other plant cysteine proteinases including greater than 70% identity with a cysteine proteinase from Arabidopsis thaliana. Amino acids in the active site of cysteine proteinases were conserved in the pDCCP1 peptide. RNA gel blot analysis revealed that the expression of pDCCP1 increased substantially with the onset of ethylene production and senescence of petals. Increased pDCCP1 expression was also associated with ethylene production in other senescing floral organs including ovaries and styles. The pDCCP1 transcript accumulated in petals treated with exogenous ethylene within 3 h and treatment of flowers with 2,5-norbornadiene, an inhibitor of ethylene action, prevented the increase in pDCCP1 expression in petals. The temporal and spatial patterns of pDCCP1 expression suggests a role for cysteine proteinase in the loss of protein during floral senescence.

Developmental and UV Light Regulation of the Snapdragon Chalcone Synthase Promoter.

Expression directed by the 1.1-kb snapdragon chalcone synthase (CHS) promoter linked to the [beta]-glucuronidase reporter gene has been studied in transgenic tobacco. The pattern of expression of the chimeric gene was compared with the expression of the endogenous CHS genes in tobacco and snapdragon. We demonstrate that expression of the CHS promoter is controlled in both an organ-specific and tissue-specific manner. The highest level of expression was observed in immature seeds. Deletions were used to define regions of the promoter required for expression in roots, stems, leaves, seeds, and flower petals of transgenic plants. We have defined the minimal sequences required for expression in different organs and mapped regions of the promoter that influence expression in either a positive or negative manner. A promoter fragment truncated to -39 activates transcription in roots of 4-week-old seedlings, whereas a fragment extending to -197 bp directs expression in petals and seeds. A positive regulatory element located between -661 and -566 and comprising a 47-bp direct repeat is active in all tissues investigated except petals. UV light-regulated expression in leaves of transgenic tobacco seedlings is dependent on the presence of sequences also required for leaf-specific expression. Within the intact promoter, sequences that individually confer different patterns of expression interact to produce the highly regulated expression pattern of CHS.

Sequence Requirements of the 5-Enolpyruvylshikimate-3-phosphate Synthase 5[prime]-Upstream Region for Tissue-Specific Expression in Flowers and Seedlings.

We have analyzed expression from deletion derivatives of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) 5[prime]-upstream region in transgenic petunia flowers and seedlings. In seedlings, expression was strongest in root cortex cells and in trichomes. High-level expression in petals and in seedling roots was conferred by large (>500 base-pair) stretches of sequence, but was lost when smaller fragments were analyzed individually. This apparent requirement for extensive sequence suggests that combinations of cis-elements that are widely separated control tissue-specific expression from the EPSPS promoter. We have also used the high-level, petal-specific expression of the EPSPS promoter to change petal color in two mutant petunia lines.