Phloem translocation of gibberellins in three species of higher plants

Phloem sap was collected from white lupin (Lupinus albus L.), cowpea (Vigna unguiculata L.) and castor bean (Ricinus communis L.) and analysed for gibberellins (GAs) using gas chromatography-mass spectrometry (GC-MS). A large number of GAs were found in the phloem exudate of all three species, particularly where the sap was collected from pods (white lupin and cowpea) and in these legumes GAs representing both the early C-13-hydroxylation and non-hydroxylation pathways of biosynthesis were identified. In the sap collected from the vegetative tissues of castor bean the number of GAs identified was fewer than that in the other species, representing mainly the non-hydroxylation pathway. Data from sap collected from the pedicel and stylar ends of pods and by making feeds of radiolabelled GAs to seeds in situ in white lupin indicate that the GAs present in the phloem are derived mainly from the vegetative tissues of the plant. No evidence for metabolism of GAs in the phloem could be found.     

Interactions Between Light and Plant Hormones During De-etiolation

The transition from a dark-grown (etiolated) to a light-grown (de-etiolated) morphology is marked by a number of dramatic phenotypic changes such as a significant reduction in the rate of shoot elongation, opening of the apical hook, expansion of true leaves and the development of mature chloroplasts. Many of these developmental processes are also known to be regulated by plant hormones. In this review we discuss the interactions between light and plant hormones and their role in mediating phenotypic change during de-etiolation. Clear evidence exists for a light-mediated reduction in gibberellin A, GA levels and response in pea, which is thought to be responsible, at least in part, for the reduction of shoot elongation during de-etiolation. Indirect evidence from a number of species has been used to suggest that the reduction in shoot elongation could also be mediated by a reduction in brassinosteroid (BR) levels. However, direct evidence recently obtained from pea and rice demonstrates that de-etiolation is not mediated, or even accompanied, by a reduction in BR levels. Ethylene is known to play an integral role in apical hook formation and maintenance in plants. However, the physiological significance of light-induced changes in IAA and ABA levels found in some species is not clear. Recent molecular data provide evidence of interactions between light-and IAA/CK-signalling pathways. Potential mechanisms for these interactions are discussed.     

Hormone levels and response during de-etiolation in pea

The objective of this study was to increase our understanding of the hormonal regulation of de-etiolation by investigating endogenous hormone levels and response in etiolated pea ( Pisum sativum L.) seedlings after exposure to continuous white light. Recent reports suggest that de-etiolation may result from the down-regulation of an enzyme in the brassinosteroid (BR) biosynthesis pathway in pea. A subsequent review highlighted the need for direct measurements of BR levels to support this hypothesis. We have shown that endogenous castasterone and 6-deoxocastasterone levels are not greatly reduced after exposure to light; indeed, 6-deoxocastasterone levels were actually increased. Similarly, the elongation response to exogenous brassinolide was greater in plants grown in continuous light, or in dark-grown plants that had been transferred into the light, than in plants that were grown in continuous darkness. These results provide further evidence to suggest that BRs do not negatively regulate de-etiolation in pea. However, changes in the levels of several other hormones have also been implicated in light-regulated development. We have simultaneously quantified indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid levels in whole seedlings, which revealed a complex pattern of changes in the levels of these substances after exposure to light. The first and most dramatic of these changes was a significant reduction in GA(1) levels, which reached a minimum 8 h after exposure to light. Whilst GA(1) levels rapidly decreased, IAA levels remained unchanged in the short term after exposure to light, suggesting that GA(1) levels may be the primary factor regulating the reduction in elongation growth during de-etiolation. In the long term after exposure to light, IAA levels underwent a transitory increase, which peaked at 48 h, and had abated by 96 h. However, abscisic acid levels remained unchanged in the first 1 h after exposure to light before undergoing a steady decline over time. The relative importance of these changes in mediating light-induced changes in plant morphology is discussed.     

Identification of a <Emphasis Type="Italic">Lotus</Emphasis> viral pathogen

A virus collection was used to identify a pathogen suitable for laboratory use with the model legume Lotus japonicus. Several Lotus species or L. japonicus accessions were tested and various degrees of susceptibility to the Arabis mosaic virus derived from barley (ArMV-ba) were found. Virus multiplication and persistence in Lotus tissue were examined, as well as plant responses to it. Sensitivity to the virus among the accessions and species is discussed in light of their geographical origin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anisocotyly and meristem initiation in an unorthodox plant, <Emphasis Type="Italic">Streptocarpus rexii</Emphasis> (Gesneriaceae)

In common with most Old World Gesneriaceae; Streptocarpus Lindl. shows anisocotylous growth, i.e., the continuous growth of one cotyledon after germination. Linked to this phenomenon is an unorthodox behaviour of the shoot apical meristem (SAM) that determines the growth pattern of acaulescent species (subgenus Streptocarpus). In contrast caulescent species develop a conventional central post-embryonic SAM (mainly subgenus Streptocarpella). We used S. rexii Lindl. as a model to investigate anisocotyly and meristem initiation in Streptocarpus by using histological techniques and analyses of the expression pattern of the meristematic marker SrSTM1 during ontogeny. In contrast to Arabidopsis thaliana (L.) Heynh., S. rexii does not establish a SAM during embryogenesis, and the first evidence of a SAM-like structure occurs during post-embryonic development on the axis (the petiolode) between the two cotyledons. The expression pattern of SrSTM1 suggests a function in maintaining cell division activity in the cotyledons before becoming localized in the basal meristem, initially at the proximal ends of both cotyledons, later at the base of the continuously growing macrocotyledon, and the groove meristem on the petiolode. The latter is equivalent to a displaced SAM seemingly originating de novo under the influence of endogenous factors. Applied cytokinin retains SrSTM1expression in the small cotyledon, thus promoting isocotyly and re-establishment of a central post-embryonic SAM. Hormone-dependent delocalization of the process of meristem development could underlie anisocotyly and the unorthodox SAM formation in Streptocarpus. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Metabolomic and genetic analyses of flavonol synthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> support the in vivo involvement of leucoanthocyanidin dioxygenase

Flavonol synthase (FLS) (EC-number 1.14.11.23), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence database accession numbers: GenBank accession EU287457 and EU287459.     

Phylogeny and comparative seed morphology of epiphytic and terrestrial species of <Emphasis Type="Italic">Liparis</Emphasis> (Orchidaceae) in Japan

To elucidate the evolution of epiphytes in Liparis section Liparis, we examined the phylogenetic relationships of 16 species by using internal transcribed spacer regions of 18S–26S nuclear ribosomal DNA (ITS) and three chloroplast DNA regions (trnS-trnG spacer, trnL with trnL-trnF spacer, and partial matK). Results showed that the epiphytic L. fujisanensis is sister to the terrestrial L. koreana and L. kumokiri, while another epiphyte, L. truncata, is sister to the terrestrial L. krameri. Therefore, the two epiphytic species evolved from terrestrial species independently in section Liparis. Comparative seed morphology revealed that the epiphytes have larger embryos than their closely related terrestrial counterparts. A similar trend toward the increase of embryo size in the two epiphytic species belonging to closely related, but distinct clades suggests that the large embryo may have an advantage in the epiphytic lifestyle. The two epiphytic species share another character state, smaller air spaces in the seed than that of closely related terrestrial species, suggesting possible low dispersibility of the epiphytes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multiple subfamilies of mariner transposable elements are present in stalk-eyed flies (Diptera: Diopsidae)

The Diopsid stalk-eyed flies are an increasingly well-studied group. Presented here is evidence of the first known transposable elements discovered in these flies. The vertumnana mariner subfamily was identified in the Diopsini tribe, but could not be amplified in species of the Sphyracephalini tribe. PCR screening with degenerate primers revealed that multiple mariner subfamilies are present within the Diopsidae. Most of the sequenced elements appear to be pseudogenes; however two subfamilies are shown to be evolving under purifying selection, raising the possibility that mariner is active in some Diopsid species. Evidence is presented of a possible horizontal transfer event involving an unknown Teleopsis species and the Tephritid fly Bactrocera neohumeralis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and characterization of a hypoallergenic ortholog of Ara h 2.01

Peanut (Arachis hypogaea L.), can elicit type I allergy becoming the most common cause of fatal food-induced anaphylactic reactions. Strict avoidance is the only effective means of dealing with this allergy. Ara h 2, a peanut seed storage protein, has been identified as the most potent peanut allergen and is recognized by approximately 90% of peanut hypersensitive individuals in the US. Because peanut has limited genetic variation, wild relatives are a good source of genetic diversity. After screening 30 Arachis duranensis accessions by EcoTILLing, we characterized five different missense mutations in ara d 2.01. None of these polymorphisms induced major conformational modifications. Nevertheless, a polymorphism in the immunodominant epitope #7 (S73T) showed a 56–99% reduction in IgE-binding activity and did not affect T cell epitopes, which must be retained for effective immunotherapy. The identification of natural hypoallergenic isoforms positively contributes to future immunological and therapeutic studies and peanut cultivar development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immunocytochemical Localisation of Glycosaminoglycan-Like Activity in the Mucus and Associated Tissues of Terrestrial Slug Species (Gastropoda: Pulmonata)

Indirect immunofluorescence assays were conducted on cryotome sections of four terrestrial slug species from three distinct phylogenetic groups, Arion ater (L.), Arion hortensis (Férussac), Tandonia (Milax) budapestensis (Hazay), and Deroceras reticulatum (Müller) using monoclonal antibodies for two glycosaminoglycans (GAGs), heparan sulphate, and chondroitin sulphate. Specific staining for a heparan sulphate-like component was demonstrated in the foot and tail regions of each species and was particularly intense in A. ater and A. hortensis, notably in the epidermis and associated mucus-like material, and in mucus-like material from the pedal gland region of the latter species. Subsequent studies with A. ater confirmed the presence of heparan-sulphate–like activity in the caudal gland duct region. No evidence of specific staining for chondroitin sulphate-like activity was found in any of the slug species. This work suggests that a specific GAG, or a group of closely related GAGs, is a common component of mucus in a range of slug species and of different types of mucus, including trail (pedal) mucus and the more viscous mucus produced by the caudal gland.     

Photomorphogenic responses to UV radiation IV: a comparative study of UVB effects on growth and pigment accumulation in etiolated and de-etiolated hypocotyls of wild-type and aurea mutant of tomato Lycopersicon esculentum Mill)

ABSTRACT

The question of how de-etiolation of tomato seedling under continuous monochromatic yellow light exerts an influence on UV radiation-induced responses has been studied. Hypocotyl extension and the accumulation of anthocyanins and UV-absorbing compounds was compared in the aurea mutant of tomato and its isogenic wild type. The data of the present paper provide evidence that, during de-etiolation of tomato seedlings, yellow light exerts its effects over seedlings responsiveness to subsequent UV irradiation through several mechanisms: 1) a significant enhancement of shorter UVB wavelength efficiency in both the genotypes; 2) the abolition of UVA -blue light-induced accumulation of UV-absorbing compounds that does not involve pnyA; 3) the disappearance of UVA-blue light-induced hypocotyl growth inhibition that does not involve phyA; 4) higher anthocyanin accumulation rate in response to UV radiation mediated by phyA. Yellow light-induced growth inhibition and accumulation of UV-absorbing compound both mediated by phyA and present only in wild type tomato, appear to be completely separate from the action of UV radiation on the same responses.     

Characterisation and expression of monosaccharide transporters in lupins, <Emphasis Type="Italic">Lupinus polyphyllus</Emphasis> and <Emphasis Type="Italic">L. albus</Emphasis>

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Mutator</Emphasis>-like elements identified in melon,Arabidopsis and rice contain ULP1 protease domains

The transposon Mutator was first identified in maize, and is one of the most active mobile elements in plants. The Arabidopsis thaliana genome contains at least 200 Mutator-like elements (MULEs), which contain the Mutator-like transposase gene, and often additional genes. We have detected a novel type of MULEs in melon (CUMULE), which, besides the transposase, contains two ubiquitin-like specific protease-like sequences (ULP1). This element is not present in the observed location in some melon cultivars. Multiple copies of this element exist in the Cucumis melo genome, and it has been detected in other Cucurbitaceae species. Analysis of the A. thaliana genome revealed more than 90 CUMULE-like elements, containing one or two Ulp1-like sequences, although no evidence of mobility exists for these elements. We detected various putative transposable elements containing ULP1-like sequences in rice. The discovery of these MULEs in melon and Arabidopsis, and the existence of similar elements in rice and maize, suggest that a proteolytic function may be important for this subset of the MULE transposable elements. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Nucleotide sequence data reported are available in the GenBank database under the accession number AY524004.     

Constitutive expression of two apple (Malus x domestica Borkh.) homolog genes of LIKE HETEROCHROMATIN PROTEIN1 affects flowering time and whole-plant growth in transgenic Arabidopsis

Fruit trees, such as apple (Malus × domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A fractionation procedure for identifying novel proteins induced by chill stress in Arabidopsis thaliana

Extraction of plant proteins using typical extraction buffers leaves insoluble debris that cannot be investigated by conventional 2-DE technologies. In this paper, we present a scalable, off-line procedure for extraction of Arabidopsis thaliana homogenates that can be used in combination with both in-gel digestion and mass spectrometry. Based on sequential NaCl gradients and strong detergent fractionation, this new strategy allowed detection of 11 novel proteins from Arabidopsis thaliana that were altered in response to chilling stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.