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Role of rhizobial lipo-chitin oligosaccharide signal molecules in root nodule organogenesis.

The role of oligosaccharide molecules in plant development is discussed. In particular the role of the rhizobial lipo-chitin oligosaccharide (LCO) signal molecules in the development of the root nodule indicates that oligosaccharides play an important role in organogenesis in plants. Recent results of the analyses of structures and of the biosynthesis of the LCO molecules are summarized in this paper. The knowledge and technologies that resulted from these studies will be important tools for further studying the function of LCO signals in the plant and in the search for analogous signal molecules produced by plants.

Carbohydrate Sequence↗

A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors.

Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants. PME-mediated changes in cell wall pectin structure play important roles in plant development. Genome sequencing projects have revealed the existence of large PME multigene families in higher plants. Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells. Several assay procedures for the determination of PME activity have been reported. However, previous protocols suffered from various limitations. Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3). This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range. Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI.

Alcohol Dehydrogenase↗

A putative rolB gene homologue of the Agrobacterium rhizogenes TR-DNA has different morphogenetic activity in tobacco than rolB.

Agrobacterium rhizogenes strains of the agropine type harbor on their Ri-plasmid two T-DNAs, a left TL-DNA and a right TR-DNA. The rolB gene of the TL-DNA is the major factor in the pathogenesis of the hairy-root disease and its constitutive expression interfere profoundly with plant morphogenesis. We have tested whether the expression of its sequence related putative homologue from the TR-DNA (rolBTR) may cause also bacterial virulence or affect plant development. Unlike rolB, rolBTR is unable to induce root formation on tobacco leaf discs. Tobacco plants expressing a chimeric 35S::rolBTR gene have reduced stature, off-shoots at the stem base and bent and wrinkled leaves with epinastic growth. 14 N-terminal amino acids which are absent in the rolB protein are indispensable to rolBTR protein activity. The characteristic tyrosine phosphatase super family motif CX5R is absent in the rolBTR protein. For rolB this motif is possibly functionally relevant. We conclude that the rolBTR gene product has morphogenic activity but is not a functional homologue of the rolB protein.

Amino Acid Sequence↗

Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters.

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H(+)/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H(+)/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.

Antiporters↗

Nitrogen Stress and Apparent Photosynthesis in Symbiotically Grown Pisum sativum L.

Pea plants (Pisum sativum L. cv. Alaska) were inoculated individually with one of 15 Rhizobium leguminosarum strains and grown under uniform environmental conditions in the absence of combined N. Differences in effectiveness of the Rhizobium strains produced plants with differing rates of whole plant apparent N(2) fixation and total N content at the same morphological stage of development. Plants were analyzed to determine interactions between N(2) fixation, N allocation, apparent photosynthesis, and growth. Total leaf N increased linearly with total N(2) fixation (R(2) = 0.994). The proportion of total N allocated to leaves, the per cent N content of individual leaves, and the photosynthetic efficiency of individual leaves showed a curvilinear response with increasing plant N content. Differences in allocation patterns of leaf N between plants with low and high N content resulted in differences in the relationship between total N content and plant dry weight. Results from this study show that N(2) fixation interacts with leaf photosynthetic efficiency and plant growth in a manner that is dependent upon the allocation of symbiotically fixed N.

Journal Article↗

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon.

The extent and occurrence of water stress-induced "patchy" CO(2) uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [(14)C]CO(2) before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to -1.1 megapascals during a 4-day period did result in heterogenous CO(2) assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO(2) assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO(2)] in stressed bean leaves. When extent of heterogenous CO(2) uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to -1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO(2) assimilation patterns when stressed to a leaf water potential of -2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.

Journal Article↗

Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants.

A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study beta-1,3-glucan synthase activity changes. All tissues contained an active beta-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active beta-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in beta-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that beta-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development.

Journal Article↗

Ectopic expression of rice OsMADS1 reveals a role in specifying the lemma and palea, grass floral organs analogous to sepals.

MADS-domain-containing transcription factors play diverse roles in plant development. The prototypic members of this gene family are the floral organ identity genes of the model dicotyledonous plant, Arabidopsis thaliana. Sequence relatedness and function ascribe them to AP1/AGL9, AG, AP3 and PI gene groups. The rice MADS-box gene, OsMADS1, is a member of the AP1/ AGL9 sub-group. Tomato and Petunia members of this sub-group specify floral meristem identity and control organ development in three inner whorls. Reported here are phylogenetic analyses that show OsMADS1 to form a distinct clade within the AGL9 gene family. This sub-group currently has only three other monocot genes. We have studied the expression pattern of OsMADS1 and determined the consequences of its ectopic expression in transgenic rice plants. OsMADS1 is not expressed during panicle branching; earliest expression is in spikelet meristems where it is excluded from the outer rudimentary/sterile glumes. During organogenesis, OsMADS1 expression is confined to the lemma and palea, with weak expression in the carpel. Ectopic OsMADS1 expression results in stunted panicles with irregularly positioned branches and spikelets. Additionally, in spikelets, the outer rudimentary glumes are transformed to lemma/palea-like organs. Together, these data suggest a distinct role for OsMADS1 and its monocot relatives in assigning lemma/palea identity.

Alleles↗

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana.

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.

Alleles↗

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

Journal Article↗

Control of the cell cycle.

Cell division is arguably the most fundamental developmental process for single-celled and multicellular organisms alike. The pathway from one cell division to the next is known as the cell cycle. A conserved biochemical regulatory network controls progress along this pathway in plants, animals, and yeasts. This review is intended to serve as a primer on the current state of the eukaryotic cell cycle regulatory model, an introduction to the special roles of cell division and its control in plant development, and a review of recent progress in applying the universal mitotic control paradigm to higher plant systems.

Amino Acid Sequence↗

A novel cold-inducible gene from Arabidopsis, RCI3, encodes a peroxidase that constitutes a component for stress tolerance.

A cDNA from Arabidopsis corresponding to a new cold-inducible gene, RCI3 (for Rare Cold Inducible gene 3), was isolated. Isoelectric focusing electrophoresis and staining of peroxidase activity demonstrated that RCI3 encodes an active cationic peroxidase. RNA-blot analysis revealed that RCI3 expression in response to low temperature is negatively regulated by light, as RCI3 transcripts were exclusively detected in etiolated seedlings and roots of adult plants. RCI3 expression was also induced in etiolated seedlings, but not in roots, exposed to dehydration, salt stress or ABA, indicating that it is subjected to a complex regulation through different signaling pathways. Analysis of transgenic plants containing RCI3::GUS fusions established that this regulation occurs at the transcriptional level during plant development, and that cold-induced RCI3 expression in roots is mainly restricted to the endodermis. Plants overexpressing RCI3 showed an increase in dehydration and salt tolerance, while antisense suppression of RCI3 expression gave dehydration- and salt-sensitive phenotypes. These results indicate that RCI3 is involved in the tolerance to both stresses in Arabidopsis, and illustrate that manipulation of RCI3 has a potential with regard to plant improvement of stress tolerance.

Abscisic Acid↗

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: self-inhibition by dimerization.

12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.

Amino Acid Sequence↗

A biotest for evaluating copper bioavailability to plants in a contaminated soil.

This work aimed at defining the optimal conditions for a novel ecotoxicological test designed for evaluating the bioavailability and phytotoxicity of metals to plants. This biotest, which provided easy access to roots, shoots, and rhizosphere soil, was applied to a vineyard calcareous soil that had been contaminated by the application of Cu fungicides. A preliminary hydroponic experiment comparing various levels of solution Cu concentration enabled us to determine the no observable adverse effects concentration (NOAEC), which was in the range 5 to 20 microM total Cu (0.01-0.06 microM free Cu ion) for rape (Brassica napus L. cv. Goeland). For the biotest, rape was grown in hydroponic conditions for 21 d in pots designed so that plants developed a planar mat of roots at the surface of a polyamide mesh. By then, the plants were transferred for 4 or 8 d onto a 1- or 3-mm-thick soil layer that was separated from the root mat by the mesh and connected to a reservoir of nutrient solution or deionized water via a filter paper wick. An 8-d period was the best option as it enabled plant growth to be significant. The use of 1-mm soil thickness was recommended if the biotest aimed at investigating root-induced changes in the rhizosphere. Although it may cause some artifacts, compared with deionized water, nutrient solution provided better standardized conditions for comparing widely differing soil samples. The studied soil did not induce any Cu phytotoxicity in spite of its fairly large total Cu content.

Biological Assay↗

Genome-wide analysis of the MADS-box gene family in Populus trichocarpa.

Populus trichocarpa (poplar) is distinguished from its herbaceous counterparts Arabidopsis and rice by its woody structure, perennial life cycle, and dioecious, two-whorled flowers. MADS-box genes are known to be involved in many important processes during plant development. Investigation of the poplar genome revealed 105 putative functional MADS-box genes and 12 pseudogenes. These numbers are comparable to those in Arabidopsis. However, poplar has 64 type II MADS-box genes, implying a higher birth rate when compared with Arabidopsis (64 vs.47). In addition to duplications for floral organ identity genes, TM3-like, StMADS11, ANR1 and Bs clades were significantly increased. As indicated by the phylogenetic analysis, there exist at least two MIKC* MADS-box genes in the poplar genome. In contrast, only 41 putative functional type I genes and 9 pseudogenes were found, suggesting that the poplar type I MADS-box genes have experienced a lower rate in both birth and death. Poplar MADS-box genes were distributed on all linkage groups (LGs), except LG XIX. Like other gene families, poplar MADS-box gene family has expanded through tandem gene duplication and segmental duplication events as demonstrated by two genomic regions where clustered MADS-box genes exhibited high similarities in the MADS-box domains. A survey of poplar EST sequences showed that MADS-box genes were expressed in wood and cambium tissues, which are specific to woody plants. The expression of common MADS-box gene in tree-specific tissues suggests that the novel function combinations of a set of genes similar to those in herbaceous plants may account for the development of woody characteristics in poplar.

Arabidopsis↗

Epiphylly in a Variant of Helianthus Annuus x H. Tuberosus Induced by In vitro Tissue culture.

A variant clone (EMB-2) derived by in vitro tissue culture of the interspecific hybrid Helianthus annuus x Helianthus tuberosus shows a particular deviation from the usual pattern of plant development in that it produces, both in vitro and in vivo, epiphyllous embryos and/or shootlike structures. Ectopic structures, which are usually arranged in clusters or rows along preexisting veins, originate asynchronously from epidermal cells of the adaxial surface of the leaf blade. Sometimes embryos and buds are also detected on the adaxial plane of the petioles and at the nodes of the stem. EMB-2 individual plants differ greatly in terms of the timing and extent of phenotypic expression of epiphylly. Leaves precociously affected by ectopic structures show a more drastic alteration in the differentiation process. Growth is arrested, the spongy parenchyma and air spaces are absent, and the mesophyll cells do not enlarge. Excluding the veins and epidermis, the leaves are wholly composed of isodiametric cells that are regularly arranged in parallel rows that have dense cytoplasm and prominent nuclei. Ectopic structures isolated from leaves and cultured in vitro mostly produce plantlets with the same phenotype as the original clones. In vivo, the EMB-2 plants are propagated by tubers. Often, the shoot-meristems that originate from tubers exhibit a teratological appearance and die without further development. However, several normal shoots grow and produce plants that display epiphyllous structures like those of the parent plants. Alterations of the endogenous hormonal levels or mutations in genes involved in the switch from indeterminate to determinate cell fate may be responsible for the ectopic development of shoots and embryos on leaves of EMB-2 variant.

Journal Article↗

Developmental regulation of intercellular protein trafficking through plasmodesmata in tobacco leaf epidermis

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.

Journal Article↗

RNA-binding proteins in plants: the tip of an iceberg?

RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.

Chloroplasts↗