4-Coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A.

BACKGROUND: Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis. RESULTS: Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae). CONCLUSIONS: Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast.

Identification and Evaluation of Aromatic Volatile Compounds in 26 Cultivars and 8 Hybrids of Freesia hybrida.

Freesia hybrida is a group of cultivars in the genus Freesia with a strong floral scent composed of diverse volatile organic compounds (VOCs). In this study, the VOCs of 34 F. hybrida were extracted and analyzed by headspace solid phase microextraction and gas chromatography mass spectrometry (HS-SPME-GC-MS). A total of 164 VOCs whose relative contents were higher than 0.05% were detected. The numbers of VOCs in all germplasms differed between 11 to 38, and the relative contents ranged from 32.39% to 94.28%, in which most germplasms were higher than 80%. Terpenoids, especially monoterpenes, were the crucial type of VOCs in most germplasms, of which linalool and D-limonene were the most frequently occurring. Principal component analysis (PCA) clearly separated samples based on whether linalool was the main component, and hierarchical clustering analysis (HCA) clustered samples into 4 groups according to the preponderant compounds linalool and (E)-ß-ocimene. Comparison of parental species and hybrids showed heterosis in three hybrids, and the inherited and novel substances suggested that monoterpene played an important role in F. hybrida floral scent. This study established a foundation for the evaluation of Freesia genetic resources, breeding for the floral aroma and promoting commercial application.

Morphological and physicochemical characterization of starches from underground stems of Trimezia juncifolia collected in different phenological stages.

In this study, starches from underground stems of Trimezia juncifolia were evaluated during dry season (DSS), wet season (WSS) and sprouting (SS). Results evidenced that drought stress did not interfere with the yield, amylose content and degree of polymerization (DP) of amylopectin. However, the extraction yield in SS was 58% lower, being observed and increase of 7.5% in the content of amylose, and 13.5% in DP values for SS amylopectin, with a predominance of A-chains. The amount of total sugar, the starch granules size as well as solubility and swelling properties varied as function of the phenological status. Also, starch granules changed from A-type polymorph in DSS and SS to a CA-type in WSS. Nevertheless, it was observed a crystallinity reduction from 56% in DSS to 37.1% in SS. In addition, thermograms evidenced the presence of amylose-lipid complexes, with endothermic transition temperatures being affected by drought stress and sprouting. Finally, results demonstrate that underground stems from T. juncifolia have adaptative strategies involving changes in the morphological and physicochemical properties of the starch granules.

The Conserved and Particular Roles of the R2R3-MYB Regulator FhPAP1 from Freesia hybrida in Flower Anthocyanin Biosynthesis.

Anthocyanin biosynthesis is mainly controlled by MYB-bHLH-WD40 (MBW) complexes that modulate the expression of anthocyanin biosynthetic genes (ABGs). The MYB regulators involved in anthocyanin biosynthesis arose early during plant evolution and thus might function divergently in different evolutionary lineages. Although the anthocyanin-promoting R2R3-MYB regulators in eudicots have been comprehensively explored, little consensus has been reached about functional discrepancies versus conservation among MYB regulators from different plant lineages. Here, we integrated transcriptome analysis, gene expression profiles, gain-of-function experiments and transient protoplast transfection assays to functionally characterize the monocot Freesia hybrida anthocyanin MYB regulator gene FhPAP1, which showed correlations with late ABGs. FhPAP1 could activate ABGs as well as TT8-clade genes FhTT8L, AtTT8 and NtAN1 when overexpressed in Freesia, Arabidopsis and tobacco, respectively. Consistently, FhPAP1 could interact with FhTT8L and FhTTG1 to form the conserved MBW complex and shared similar target genes with its orthologs from Arabidopsis. Most prominently, FhPAP1 displayed higher transactivation capacity than its homologs in Arabidopsis and tobacco, which was instantiated in its powerful regulation on ABGs. Moreover, we found that FhPAP1 might be the selected gene during the domestication and rapid evolution of the wild Freesia species to generate intensive flower pigmentation. These results showed that while the MBW complex was highly evolutionarily conserved between tested monocot and core eudicot plants, participating MYB regulators showed functional differences in transactivation capacity according to their activation domain and played important roles in the flower coloration domestication and evolution of angiosperms.

A functional homologue of Arabidopsis TTG1 from Freesia interacts with bHLH proteins to regulate anthocyanin and proanthocyanidin biosynthesis in both Freesia hybrida and Arabidopsis thaliana.

The MBW complex, consisting of MYB, basic helix-loop-helix (bHLH) and WD40 proteins, regulates multiple traits in plants, such as anthocyanin and proanthocyanidin biosynthesis and cell fate determination. The complex has been widely identified in dicot plants, whereas few studies are concentrated on monocot plants which are of crucial importance to decipher its functional diversities among angiosperms during evolution. In present study, a WD40 gene from Freesia hybrida, designated as FhTTG1, was cloned and functionally characterized. Real-time PCR analysis indicated that it was expressed synchronously with the accumulation of both proanthocyanidins and anthocyanins in Freesia flowers. Transient protoplast transfection and biomolecular fluorescence complementation (BiFC) assays demonstrated that FhTTG1 could interact with FhbHLH proteins (FhTT8L and FhGL3L) to constitute the MBW complex. Moreover, the transportation of FhTTG1 to nucleus was found to rely on FhbHLH factors. Outstandingly, FhTTG1 could highly activate the anthocyanin or proanthocyanidin biosynthesis related gene promoters when co-transfected with MYB and bHLH partners, implying that FhTTG1 functioned as a member of MBW complex to control the anthocyanin or proanthocyanidin biosynthesis in Freesia hybrida. Further ectopic expression assays in Arabidopsis ttg1-1 showed the defective phenotypes of ttg1-1 were partially restored. Molecular biological assays validated FhTTG1 might interact with the endogenous bHLH factors to up-regulate genes responsible for anthocyanin and proanthocyanidin biosynthesis and trichome formation, indicating that FhTTG1 might perform exchangeable roles with AtTTG1. These results will not only contribute to the characterization of FhTTG1 in Freesia but also shed light on the establishment of flavonoid regulatory system in monocot plants, especially in Freesia hybrida.

Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida.

The development of flower scents was a crucial event in biological evolution, providing olfactory signals by which plants can attract pollinators. In this study, bioinformatics, metabolomics, and biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of volatile components in two cultivars of Freesia x hybrida, Red River® and Ambiance, which release different categories of compounds. We found that terpene synthase (TPS) genes were the pivotal genes determining spatiotemporal release of volatile compounds in both cultivars. Eight FhTPS genes were isolated and six were found to be functional: FhTPS1 was a single-product enzyme catalyzing the formation of linalool, whereas the other four FhTPS proteins were multi-product enzymes, among which FhTPS4, FhTPS6, and FhTPS7 could recognize geranyl diphosphate and farnesyl diphosphate simultaneously. The FhTPS enzymatic products closely matched the volatile terpenes emitted from flowers, and significant correlations were found between release of volatile terpenes and FhTPS gene expression. Graphical models based on these results are proposed that summarize the biosynthesis of Freesia floral volatile terpenes. The characterization of FhTPS genes paves the way to decipher their roles in the speciation and fitness of Freesia, and this knowledge could also be used to introduce or enhance scent in other plants.

Integrated micro-biochemical approach for phytoremediation of cadmium and lead contaminated soils using Gladiolus grandiflorus L cut flower.

The potential of vermicompost, elemental sulphur, Thiobacillus thiooxidans and Pseudomonas putida for phytoremediation is well known individually but their integrated approach has not been discovered so far. The present work highlights the consideration of so far overlooked aspects of their integrated treatment by growing the ornamental plant, Gladiolus grandiflorus L in uncontaminated and sewage-contaminated soils (sulphur-deficient alluvial Entisols, pH 7.6-7.8) for phytoremediation of cadmium and lead under pot experiment. Between vermicompost and elemental sulphur, the response of vermicompost was higher towards improvement in the biometric parameters of plants, whereas the response of elemental sulphur was higher towards enhanced bioaccumulation of heavy metals under soils. The integrated treatment (T7: vermicompost 6g and elemental sulphur 0.5gkg(-1) soil and co-inoculation of the plant with T. thiooxidans and P. putida) was found superior in promoting root length, plant height and dry biomass of the plant. The treatment T7 caused enhanced accumulation of Cd up to 6.96 and 6.45mgkg(-1) and Pb up to 22.6 and 19.9mgkg(-1) in corm and shoot, respectively at the contaminated soil. T7 showed maximum remediation efficiency of 0.46% and 0.19% and bioaccumulation factor of 2.92 and 1.21 and uptake of 6.75 and 21.4mgkg(-1) dry biomass for Cd and Pb respectively in the contaminated soil. The integrated treatment T7 was found significant over the individual treatments to promote plant growth and enhance phytoremediation. Hence, authors conclude to integrate vermicompost, elemental sulphur and microbial co-inoculation for the enhanced clean-up of Cd and Pb-contaminated soils.

Cadmium translocation by contractile roots differs from that in regular, non-contractile roots.

BACKGROUND AND AIMS: Contractile roots are known and studied mainly in connection with the process of shrinkage of their basal parts, which acts to pull the shoot of the plant deeper into the ground. Previous studies have shown that the specific structure of these roots results in more intensive water uptake at the base, which is in contrast to regular root types. The purpose of this study was to find out whether the basal parts of contractile roots are also more active in translocation of cadmium to the shoot. METHODS: Plants of the South African ornamental species Tritonia gladiolaris were cultivated in vitro for 2 months, at which point they possessed well-developed contractile roots. They were then transferred to Petri dishes with horizontally separated compartments of agar containing 50 µmol Cd(NO3)2 in the region of the root base or the root apex. Seedlings of 4-d-old maize (Zea mays) plants, which do not possess contractile roots, were also transferred to similar Petri dishes. The concentrations of Cd in the leaves of the plants were compared after 10 d of cultivation. Anatomical analyses of Tritonia roots were performed using appropriately stained freehand cross-sections. KEY RESULTS: The process of contraction required specific anatomical adaptation of the root base in Tritonia, with less lignified and less suberized tissues in comparison with the subapical part of the root. These unusual developmental characteristics were accompanied by more intensive translocation of Cd ions from the basal part of contractile roots to the leaves than from the apical-subapical root parts. The opposite effects were seen in the non-contractile roots of maize, with higher uptake and transport by the apical parts of the root and lower uptake and transport by the basal part. CONCLUSIONS: The specific characteristics of contractile roots may have a significant impact on the uptake of ions, including toxic metals from the soil surface layers. This may be important for plant nutrition, for example in the uptake of nutrients from upper soil layers, which are richer in humus in otherwise nutrient-poor soils, and also has implications for the uptake of surface-soil pollutants.

Irisflorentin modifies properties of mouse bone marrow-derived dendritic cells and reduces the allergic contact hypersensitivity responses.

Irisflorentin is an isoflavone component derived from the roots of Belamcanda chinensis (L.) DC. In traditional Chinese medicine, this herb has pharmacological properties to treat inflammatory disorders. Dendritic cells (DCs) are crucial modulators for the development of optimal T-cell immunity and maintenance of tolerance. Aberrant activation of DCs can induce harmful immune responses, and so agents that effectively improve DC properties have great clinical value. We herein investigated the effects of irisflorentin on lipopolysaccharide (LPS)-stimulated maturation of mouse bone marrow-derived DCs in vitro and in the contact hypersensitivity response (CHSR) in vivo. Our results demonstrated that treatment with up to 40 µM irisflorentin does not cause cellular toxicity. Irisflorentin significantly lessened the proinflammatory cytokine production (tumor necrosis factor-α, interleukin-6, and interleukin-12p70) by LPS-stimulated DCs. Irisflorentin also inhibited the expression of LPS-induced major histocompatibility complex class II and costimulatory molecules (CD40 and CD86) on LPS-stimulated DCs. In addition, irisflorentin diminished LPS-stimulated DC-elicited allogeneic T-cell proliferation. Furthermore, irisflorentin significantly interfered with LPS-induced activation of IκB kinase, c-Jun N-terminal kinase, and p38, as well as the nuclear translocation of NF-κB p65. Subsequently, treatment with irisflorentin obviously weakened 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity. These findings suggest new insights into the role of irisflorentin as an immunotherapeutic adjuvant through its capability to modulate the properties of DCs.

Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus.

Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus.

Wild-boar disturbance increases nutrient and C stores of geophytes in subalpine grasslands.

PREMISE OF THE STUDY: Wild-boar soil disturbance (i.e., rooting) increases the abundance of some species of geophytes (i.e., plants with underground renewal buds) in upland meadows. However, the mechanisms that could lead to such enhanced prevalence remain unexplored. • METHODS: We analyzed the effects of wild-boar disturbance on the size, nutrient (N, P, K, C, and total ash), and nonstructural carbohydrate (soluble sugars, starch plus fructans, and total nonstructural carbohydrate) content of the storage organs of five taxa of upland geophytes. Results were explored in relation to the nutrient availability (total N, available P, and K) in the soil. • KEY RESULTS: Wild-boar rooting increased the size and the nutrient content of the storage organs of geophytes. Such enhanced storage was further promoted by rooting recurrence and intensity. Although we could not detect a direct impact of rooting on soil nutrient concentrations, plants were clearly N limited and such limitation was ameliorated in areas rooted by wild boar. Furthermore, plant-soil interactions for N were different in rooted areas, where plant N-concentrations responded positively to soil N. • CONCLUSIONS: Geophytes growing in rooted areas have an increased nutrient value, which may promote the revisit of wild boars to previously rooted areas, with further positive feed-back effects on plant quality. This plant-animal interaction may shape upland geophyte communities.

Floral rewards in the tribe Sisyrinchieae (Iridaceae): oil as an alternative to pollen and nectar?

Iridaceae is one of the few families in which floral oils are produced and collected by pollinators as a resource. Perigonal nectaries and trichomal elaiophores are highly unusual within the tribe Sisyrinchieae. Both structures occur mainly on the staminal column, while they are usually distributed on the tepals in the other tribes of the subfamily Iridoideae. Sisyrinchieae is the largest tribe of Iridaceae present on the American continent, and the diversity observed may be related to the exceptional development of trichomal elaiophores within the genus Sisyrinchium, but knowledge concerning the other types of nuptial glandular structures within the tribe is still limited, preventing us from estimating their implication for species diversity. Structural observations and histochemical tests were performed to identify and characterize glandular structures and pollen rewards within the flowers of the genera Orthrosanthus, Sisyrinchium and Solenomelus. Perigonal nectaries were detected only in Solenomelus segethi, and trichomal elaiophores were characterized only within Sisyrinchium. All species showed large amounts of additional resources available for pollinators in the form of pollenkitt and polysaccharides present in the cytoplasm of the pollen grains. The results are discussed in a phylogenetic context, with regard to pollinators and floral rewards reported for the tribe Sisyrinchieae.

The 5'UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis.

BACKGROUND: There are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for ß-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter. RESULTS: Gladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5' GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5'UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5'UTR-intron. The 5'-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis. CONCLUSIONS: The GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5'UTR-intron from GUBQ1 resulted in a similar pattern of ß-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.

Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus.

BACKGROUND AND AIMS: Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined. METHODS: Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species. KEY RESULTS AND CONCLUSIONS: Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA.

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.

Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: a deeper study in Crocus sativus and its allies.

The plastoglobule-targeted enzyme carotenoid cleavage dioxygenase (CCD4) mediates the formation of volatile C13 ketones, such as ß-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic carotenoids. Here, we report the isolation and analysis of CCD4 genomic DNA regions in Crocus sativus. Different CCD4 alleles have been identified: CsCCD4a which is found with and without an intron and CsCCD4b that showed the presence of a unique intron. The presence of different CCD4 alleles was also observed in other Crocus species. Furthermore, comparison of the locations of CCD4 introns within the coding region with CCD4 genes from other plant species suggests that independent gain/losses have occurred. The comparison of the promoter region of CsCCD4a and CsCCD4b with available CCD4 gene promoters from other plant species highlighted the conservation of cis-elements involved in light response, heat stress, as well as the absence and unique presence of cis-elements involved in circadian regulation and low temperature responses, respectively. Functional characterization of the Crocus sativus CCD4a promoter using Arabidopsis plants stably transformed with a DNA fragment of 1400 base pairs (P-CsCCD4a) fused to the ß-glucuronidase (GUS) reporter gene showed that this sequence was sufficient to drive GUS expression in the flower, in particular high levels were detected in pollen.

Hormonal and cell division analyses in Watsonia lepida seedlings.

The regeneration ability, cell division activity, auxin and cytokinin content of seedling regions and hypocotyl subsections of Watsonia lepida were studied. A total of 21 different cytokinins or conjugates were found in seedlings, with the highest cytokinin content in meristematic regions (root and shoot apical meristems). The greatest contribution to the cytokinin pool came from the biologically inactive cZRMP, suggesting that significant de novo synthesis was occurring. Five different auxins or conjugates were detected, being concentrated largely in the shoot apical meristem and leaves, IAA being the most abundant. Analysis of hypocotyl subsections (C1-C4) revealed that cell division was highest in subsection C2, although regeneration in vitro was significantly lower than in subsection C1. Anatomically, subsection C1 contains the apical meristem, and hence has meristematic cells that are developmentally plastic. In contrast, subsection C2 has cells that have recently exited the meristem and are differentiating. Despite high rates of cell division, cells in subsection C2 appear no longer able to respond to cues that promote proliferation in vitro. Auxin and cytokinin analyses of these subsections were conducted. Possibly, a lower overall cytokinin content, and in particular the free-base cytokinins, could account for this observed difference.

Extraction of cadmium and tolerance of three annual cut flowers on Cd-contaminated soils.

To evaluate the production potential and Cd removal by three flower crops, viz.: marigold (Tagetes erecta), chrysanthemum (Chrysanthemum indicum) and gladiolus (Gladiolus grandiflorus), an experiment was conducted on differentially contaminated soils (DTPA-Cd 0.6-68.4 mg kg(-1)). Biotoxicity of Cd lead to reductions in growth and flower yield of marigold at DTPA-Cd >or= 7.9 mg kg(-1) soil, while the productivity of chrysanthemum and gladiolus was sustained up to 21.2 mg kg(-1). DTPA-Cd for 50% yield reduction (C(50)) was 85, 106 and 215 mg kg(-1) soil for marigold, chrysanthemum and gladiolus, respectively, that indicates a better Cd-tolerance in gladiolus. The uptake of Cd increased with contents in soils and the maximum accumulation occurred in leaves. Among the economic parts, gladiolus spikes accumulated the highest Cd (7.2) followed by flowers of marigold (6.5) and chrysanthemum (4.0 mg kg(-1)). But, because of higher biomass, the total Cd removal was the maximum with chrysanthemum (8.3) followed by gladiolus (6.0) and the minimum (2.6 mg m(-2)) with marigold. Gladiolus with highest tolerance and Cd-content in saleable part holds potential to clean up the moderately contaminated soils.

A novel ethylene receptor homolog gene isolated from ethylene-insensitive flowers of gladiolus (Gladiolus grandiflora hort.).

Gladiolus is an ethylene insensitive flower whose exogenous ethylene and ethylene inhibitors have no effect on the petal senescence process. To study which processes in gladiolus are associated with changes in ethylene perception, two types of gladiolus genes, named GgERS1a and GgERS1b, respectively, homologous to the Arabidopsis ethylene receptor gene ERS1 were isolated. GgERS1a is conserved in terms of exon numbers and intron positions, whereas GgERS1b is almost same with GgERS1a except lacking 636 nucleotide encoding first and second histidine kinase (HisKA) motifs. The sequence data on full length genomic DNA indicated that both GgERS1a and b were spliced from different genomic DNA. As the result of mRNA expression study, in spite of lacking the two significant motifs, the expression of GgERS1b dramatically changed with advance in petal senescence, whereas the level of GgERS1a expressed highly and constitutively. The result suggests that both the genes possess a significant role for the subfunctionalization process to provide ethylene insensitivity in gladiolus flowers.

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.