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PubMed1999

The HD-Zip gene ATHB6 in Arabidopsis is expressed in developing leaves, roots and carpels and up-regulated by water deficit conditions.

Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors as yet found only in plants. We have characterized one HD-Zip gene, ATHB6, from Arabidopsis thaliana. ATHB6 was expressed constitutively in seedlings, but significantly up-regulated in seedlings subjected to water deficit, osmotic stress or exogenous treatment with abscisic acid (ABA), an induction being detectable within 30 min. The ATHB6 induction was impaired in the two ABA-insensitive mutants, abi1 and abi2, but unaffected in the abi3 mutation. The induction was ABA-dependent, since no increase in ATHB6 transcript was detected in the ABA-deficient mutant aba-3 subjected to drought treatment. These results suggest that ATHB6 may act downstream to both ABI1 and ABI2 in a signal transduction pathway mediating a drought stress response. A translational fusion of the ATHB6 promoter with the reporter gene GUS (ATHB6::GUS) in transgenic A. thaliana plants showed high-level expression in leaf primordia. Expression in developing cotyledons, leaves, roots and carpels was restricted to regions of cell division and/or differentiation. The expression in the cotyledons was detectable in the epidermis and high in the stomatal cells. In mature cotyledons and leaves the marker gene was expressed only in the vascular tissue. These expression data suggest ATHB6 to have a function related to cell division and/or differentiation in developing organs.

Abscisic Acid↗
PubMed2000

Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata.

Proteinase inhibitors (PIs) of the potato type II family have been identified in a number of solanaceous species. Most family members have two PI domains which are specific for either chymotrypsin or trypsin. More recently family members have been described with three or six repeated PI domains. Here we describe a novel four-domain family member produced in the stigmas and leaves of the ornamental tobacco, Nicotiana alata, which has high sequence identity with a six-domain member from the same species. Both proteins are produced as precursors that enter the secretory pathway and are subsequently processed into a series of 6 kDa Pis. The four- and six-domain precursor proteins were isolated from immature stigmas and characterised by mass spectrometry which revealed that both proteins had been trimmed at the N-terminus, at a position corresponding to the predicted signal peptide cleavage site. Furthermore, no post-translational modifications were apparent.

Amino Acid Sequence↗
PubMed2000

Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment.

The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.

Alcohol Dehydrogenase↗
PubMed2000

Characterization of Ricinus communis phloem profilin, RcPRO1.

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.

Actins↗
PubMed2000

Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.).

A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils. Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs. OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants. The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice. The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding. The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate. Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family. The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated. The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis. The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation. The flower-predominant gene expression was identified also in the transgenic rice plants. The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants. The possible functions of OsChia 1;175 are discussed.

Amino Acid Sequence↗
PubMed2001

Immunocytochemical location of endogenous cytokinins in buds of kiwifruit (Actinidia deliciosa) during the first hours of in vitro culture.

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase as markers for light microscopy or immunoglobulin G-gold for electron microscopy respectively were used for the localization of cytokinins [9-beta-D-ribofuranosil-N6-(delta2-isopentenil) adenina ([9R]iP), 9-beta-D-ribofuranosyl-zeatin ([9R]Z) and 9-beta-D-ribofuranosyl-dihydrozeatin ([9R](diH)Z)] in kiwifruit (Actinidia deliciosa) meristematic cells of the second nodal segment. Immunolocation at the cellular level was carried out in cells from explants grown during 16 and 72 h in liquid medium. Subcellular immunolocalization was performed in cells from explants grown for 35 d on agar solidified-medium and for 30 min, 4 and 16 h in liquid medium with cellulose plugs as explant support. Taken as a whole, the results obtained for Actinidia deliciosa show that the studied cytokinins change their location during the culture period, although they can always be found to a greater or lesser extent in the nucleus and the cytoplasm. For instance, [9R]Z appears in the cytoplasm and in the nucleus during the first hours of culture and later is the only one that appears located mainly in nucleus. On the other hand, [9R](diH)Z changes from being predominantly located in the nucleus to practically appearing only in the cytoplasm at the end of the culture period. [9R]iP is principally found up to 4 h of culture in the cytoplasm, and at 16 h is evenly distributed in all the subcellular compartments except in the chloroplast. The existence of a large amount of cytokinins in the nucleus during the first hours of culture compared with the immunolabelling density at 35 d is probably due to the activation of cell cycle mechanisms leading to organogenic development at the beginning of culture.

Actinidia↗
PubMed2002

Spatial and temporal expression of the orchid floral homeotic gene DOMADS1 is mediated by its upstream regulatory regions.

The orchid floral homeotic gene, DOMADSI, is a marker gene specifically expressed in the transitional shoot apical meristem during floral transition in Dendrobium Madame Thong-In. DOMADSI is not detectable in vegetative tissues except a weak expression in the stem. Its transcript is uniformly localized in both of the inflorescence meristem and floral primordia, and later expressed in almost all of the floral organs. We isolated and sequenced a 3.5 kb DOMADSI promoter fragment upstream of the transcription start site, demonstrating the location of several putative DNA-binding sites, through which MADS-box and class I knox genes may modulate the DOMADSI expression. To gain insight into the molecular basis of the regulation of DOMADS1, deletion analysis of the DOMADSI::beta-glucuronidase (GUS) gene fusions was performed by means of the stable orchid transformation systems. The study shows that the full-length upstream promoter sequence confers the same spatial and temporal GUS staining pattern as that of the distribution of DOMADSI RNA during orchid development. We also identified the distinct cis-acting regulatory regions required for the control of DOMADS1 expression in vegetative and reproductive tissues, as well as the shoot apical meristem during floral transition.

5' Flanking Region↗
PubMed2002

Cloning of the promoter for a novel barley gene, Lem1, and its organ-specific promotion of Gfp expression in lemma and palea.

The differential display method was used to identify a novel barley gene, Lem1, expressed primarily in the outer organs (lemma and palea) that enclose developing florets and seeds. The promoter was isolated from a BAC genomic clone and used in a translational fusion with a green fluorescent protein gene (Gfp) to produce a transient expression vector. After particle bombardment, Gfp was expressed only in lemmas, paleas and awns of developing spikelets. Lem1 did not promote Gfp expression in vegetative leaves or in mature spikes, although expression of co-bombarded uidA (GUS) occurred under the regulation of a ubiquitin promoter. This reproduced the developmentally regulated pattern of mRNA accumulation. Deletion studies showed that the promoter activity is confined to a cis element within 80 bp of the transcription start site. Upstream from this, the promoter contains putative auxin-, ethylene- and gibberellin-responsive elements or homologues. Lem1 was found to be a single intronless gene encoding an acidic 102 amino acid protein, possibly associated with membranes. In a two-rowed barley, Lem1 mRNA was absent in the lateral spikelets, which fail to develop, and present only in the developing median spikelets. This suggests that Lem1 may play a role in organ development.

Amino Acid Sequence↗
PubMed2002

Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage.

We isolated a stamen-specific cDNA, BSD1 (Brassica stamen specific plant defensin 1) that encodes a novel plant defensin peptide in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant defensins are antimicrobial peptides containing eight highly conserved cysteine residues linked by disulfide bridges. In BSD1, the eight cysteine residues and a glutamate residue at position 29 are conserved whereas other amino acid residues of the plant defensins consensus sequence are substituted. BSD1 transcripts accumulate specifically in the stamen of developing flowers and its level drops as the flowers mature. The recombinant BSD1 produced in Escherichia coli showed antifungal activity against several phytopathogenic fungi. Furthermore, constitutive over-expression of the BSD1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants.

Amino Acid Sequence↗
PubMed2002

Changes in the glucosinolate-myrosinase defense system in Brassica juncea cotyledons during seedling development.

Optimal defense theory (ODT) predicts that plant defenses will be allocated to plant organs and tissues in proportion to their relative fitness values and susceptibilities to attack. This study was designed to test ODT predictions on the myrosinase-glucosinolate defense system in Brassica juncea by examining the relationships between the fitness value of B. juncea cotyledons and the levels and effectiveness of cotyledon defenses. Specifically, we estimated fitness value of cotyledons during plant development by measuring plant growth and seed production after cotyledon damage or removal at successive seedling ages. Cotyledon removal within five days of emergence had a significant impact on growth and seed production, but cotyledon removal at later stages did not. Consistent with ODT, glucosinolate and myrosinase levels in cotyledons also declined with seedling age, as did relative defenses against a generalist herbivore, Spodoptera eridania, as estimated by bioassay. Declines in glucosinolates were as predicted by a passive, allometric dilution model based on cotyledon expansion. Declines in myrosinase activity were significantly more gradual than predicted by allometric dilution, suggesting active retention of myrosinase activity as young cotyledons expand.

Adaptation, Physiological↗
PubMed2002

The vascular expression pattern directed by the Eucalyptus gunnii cinnamyl alcohol dehydrogenase EgCAD2 promoter is conserved among woody and herbaceous plant species.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the last step in the synthesis of the monomeric precursors of lignin. Here, we demonstrate that the vascular expression pattern conferred by the Eucalyptus gunnii EgCAD2 promoter in transgenic poplar (Populus tremula x Populus alba) is conserved in another perennial woody angiosperm of economic interest (Vitis vinifera L.), as well as in a model herbaceous plant (Nicotiana tabacum L.). Furthermore, promoter deletion analysis performed in both tobacco and poplar allowed us to identify the proximal region [-340/-124] as essential for vascular cambium/xylem-specific expression whereas the [-124/+117] region was shown to contain cis element-driving activity in phloem fibres. Interestingly, the [-340/-124] fragment contains an AC-rich cis-acting element present in numerous genes of the phenylpropanoid pathway expressed in xylem tissues, and known as a consensus Myb transcription factor binding site, suggesting that common Myb sites may provide a mechanism by which different steps of phenylpropanoid metabolism are coordinately regulated and expressed in vascular tissues. We have also shown in both tobacco and poplar that the EgCAD2 promoter is inducible by wounding and the cis-elements responsible for wounding responsiveness are located in the distal promoter region. Taken together, our data suggest that the mechanisms controlling developmental and wounding inducible expression of the EgCAD2 promoter are conserved among perennial woody and annual herbaceous plant species enabling us now to investigate in depth the transcriptional regulation of the EgCAD2 promoter in tobacco.

Alcohol Oxidoreductases↗
PubMed2003

The promoter of a metallothionein-like gene from the tropical tree Casuarina glauca is active in both annual dicotyledonous and monocotyledonous plants.

A chimeric gene consisting of the beta-glucuronidase (gusA) reporter gene under the control of the metallothionein-like promoter cgMT1 from the tropical tree Casuarina glauca was introduced into Nicotiana tabacum via Agrobacterium tumefaciens and into Oryza sativa by particle bombardment. The strongest histochemical staining for GUS activity was observed in the root system of the transgenic plants, and especially in lateral roots. In contrast, a relatively low level of reporter gene expression was seen in the aerial tissues and GUS staining was located mainly in the plant vascular system. The average ratio of GUS activity between root and leaf was found to be 13:1 in tobacco and 1.5:1 in rice. The pattern of cgMT1 promoter activity in floral organs was found to be different in tobacco and rice. High levels of gusA gene expression were detected in the ovules, pollen grains and tapetum, whereas in rice PcgMT1 directs expression to the vascular system of the floral organs. These results suggest that PcgMT1 is potentially useful in molecular breeding to express genes of interest whose products are preferentially needed in roots.

Cotyledon↗
PubMed2003

Changes in gene expression in the wood-forming tissue of transgenic hybrid aspen with increased secondary growth.

Transgenic lines of hybrid aspen with elevated levels of gibberellin (GA) show greatly increased numbers of xylem fibres and increases in xylem fibre length. These plants therefore provide excellent models for studying secondary growth. We have used cDNA microarry analysis to investigate how gene transcription in the developing xylem is affected by GA-induced growth. A recent investigation has shown that genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under developmental-stage-specific transcriptional control. The present study shows that the highest transcript changes in our transgenic trees occurs in genes generally restricted to the early stages of xylogenesis, including cell division, early expansion and late expansion. The results reveal genes among those arrayed that are up-regulated with an increased xylem production, thus indicating key components in the production of wood.

Cell Wall↗
PubMed2003

Physcomitrella patens as a model for the study of chloroplast protein transport: conserved machineries between vascular and non-vascular plants.

A single general import pathway in vascular plants mediates the transport of precursor proteins across the two membranes of the chloroplast envelope, and at least four pathways are responsible for thylakoid protein targeting. While the transport systems in the thylakoid are related to bacterial secretion systems, the envelope machinery is thought to have arisen with the endosymbiotic event and to be derived, at least in part, from proteins present in the original endosymbiont. Recently the moss Physcomitrella patens has gained worldwide attention for its ability to undergo homologous recombination in the nuclear genome at rates unseen in any other land plants. Because of this, we were interested to know whether it would be a useful model system for studying chloroplast protein transport. We searched the large database of P. patens expressed sequence tags for chloroplast transport components and found many putative homologues. We obtained full-length sequences for homologues of three Toc components from moss. To our knowledge, this is the first sequence information for these proteins from non-vascular plants. In addition to identifying components of the transport machinery from moss, we isolated plastids and tested their activity in protein import assays. Our data indicate that moss and pea (Pisum sativum) plastid transport systems are functionally similar. These findings identify P. patens as a potentially useful tool for combining genetic and biochemical approaches for the study of chloroplast protein targeting.

Bryopsida↗
PubMed2000

The S receptor kinase determines self-incompatibility in Brassica stigma.

The self-incompatibility possessed by Brassica is an intraspecific reproductive barrier by which the stigma rejects self-pollen but accepts non-self-pollen for fertilization. The molecular/biochemical bases of recognition and rejection have been intensively studied. Self-incompatibility in Brassica is sporophytically controlled by the polymorphic S locus. Two tightly linked polymorphic genes at the S locus, S receptor kinase gene (SRK) and S locus glycoprotein gene (SLG), are specifically expressed in the papillar cells of the stigma, and analyses of self-compatible lines of Brassica have suggested that together they control stigma function in self-incompatibility interactions. Here we show, by transforming self-incompatible plants of Brassica rapa with an SRK28 and an SLG28 transgene separately, that expression of SRK28 alone, but not SLG28 alone, conferred the ability to reject self (S28)-pollen on the transgenic plants. We also show that the ability of SRK28 to reject S28 pollen was enhanced by SLG28. We conclude that SRK alone determines S haplotype specificity of the stigma, and that SLG acts to promote a full manifestation of the self-incompatibility response.

Brassica↗
PubMed2000

JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development.

Abscission is a universal and dynamic process in plants whereby organs such as leaves, flowers and fruit are shed, both during normal development, and in response to tissue damage and stress. Shedding occurs by separation of cells in anatomically distinct regions of the plant, called abscission zones (AZs). During abscission, the plant hormone ethylene stimulates cells to produce enzymes that degrade the middle lamella between cells in the AZ. The physiology and regulation of abscission at fully developed AZs is well known, but the molecular biology underlying their development is not. Here we report the first isolation of a gene directly involved in the development of a functional plant AZ. Tomato plants with the jointless mutation fail to develop AZs on their pedicels and so abscission of flowers or fruit does not occur normally. We identify JOINTLESS as a new MADS-box gene in a distinct phylogenetic clade separate from those functioning in floral organs. We propose that a deletion in JOINTLESS accounts for the failure of activation of pedicel AZ development in jointless tomato plants.

Cloning, Molecular↗
PubMed2001

Complexes of MADS-box proteins are sufficient to convert leaves into floral organs.

Genetic studies, using floral homeotic mutants, have led to the ABC model of flower development. This model proposes that the combinatorial action of three sets of genes, the A, B and C function genes, specify the four floral organs (sepals, petals, stamens and carpels) in the concentric floral whorls. However, attempts to convert vegetative organs into floral organs by altering the expression of ABC genes have been unsuccessful. Here we show that the class B proteins of Arabidopsis, PISTILLATA (PI) and APETALA3 (AP3), interact with APETALA1 (AP1, a class A protein) and SEPALLATA3 (SEP3, previously AGL9), and with AGAMOUS (AG, a class C protein) through SEP3. We also show that vegetative leaves of triply transgenic plants, 35S::PI;35S::AP3;35S::AP1 or 35S::PI;35S::AP3;35S::SEP3, are transformed into petaloid organs and that those of 35S::PI;35S::AP3;35S::SEP3;35S::AG are transformed into staminoid organs. Our findings indicate that the formation of ternary and quaternary complexes of ABC proteins may be the molecular basis of the ABC model, and that the flower-specific expression of SEP3 restricts the action of the ABC genes to the flower.

Arabidopsis↗