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Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.

Acyltransferases↗

Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog.

Mago Nashi, a protein initially shown to be essential in the development of the Drosophila oocyte, is highly conserved among species and shows no homology to any other known cellular proteins. Here we report the nucleotide sequence of a cDNA and a partial gene that encode rice Mago Nashi protein homologs. In addition, we present the tissue-specific expression pattern of mago nashi at the level of RNA and protein. The rice Mago Nashi protein shares at least 73% amino acid identity with all known animal homologs. Genomic DNA gel blot analysis indicates that two copies of the mago nashi gene exist in the rice genome, one of which has identical intron positions to those found in an Arabidopsis homolog. mago nashi is expressed in root, leaf and developing seed tissue as determined by RNA and protein gel blot analysis. Evidence from Drosophila, Caenorhabditis elegans and human studies of Mago Nashi suggests that a major function of this protein is its involvement in RNA localization. The highly conserved amino acid sequence of all Mago Nashi protein homologs across kingdoms suggests that the plant version of this protein may similarly be involved in RNA localization.

Amino Acid Sequence↗

Expression analysis and physical mapping of low-molecular-weight glutenin loci in hexaploid wheat (Triticum aestivum L.).

Gliadins and glutenins are storage proteins important in determining the bread-, noodle-, and pasta-making quality of wheat. Glutenins consist of HMW and LMW subunits. The Glu-A3, Glu-B3, and Glu-D3 loci on the short arms of chromosomes 1A, 1B, and 1D, respectively, are the major loci for LMW glutenins. To construct physical maps of the Glu-3 loci, a set of 24 high-density filters representing a 3.1x genome coverage hexaploid wheat BAC library was screened by hybridization using a probe made of 3 LMW glutenin sequences. After 2 rounds of hybridization, a subset of 536 BAC clones were selected and fingerprinted. Three developing seed cDNA libraries were also constructed. A total of 5000-6000 ESTs were generated from each library, assembled into contigs and searched by homology for LMW glutenin sequences. In total, 90 full-length LMW glutenin sequences were found to cluster into 8 distinct groups representing at least 21 different LMW glutenin subunits. A set of 24 pairs of PCR primers was designed from these groups and used as markers on the BAC clones. The combined fingerprinting and marker data were used to build the physical maps using FPC software. A total of 91 contigs comprising 254 clones were obtained and 282 clones remained singletons.

Chromosomes, Artificial, Bacterial↗

Molecular mapping of the shrunken endosperm genes seg8 and sex1 in barley (Hordeum vulgare L.).

A number of mutations affecting seed development in barley (Hordeum vulgare L.) have been known for many years; however, to date, no research has been reported that elucidates the molecular structure of the causal genes. As a first step, we initiated the linkage mapping of the two shrunken endosperm genes seg8 and sex1 using microsatellite markers. The recessive gene seg8 was mapped in the centromeric region of chromosome 7H to a 4.6 cM interval flanked by markers GBM1516 and Bmag341. The recessive sex1 gene showed xenia effects and was located in the centromeric region of barley chromosome 6H, which is in accordance to the previously reported chromosomal location in the classical linkage map. It was flanked by markers GBM5012 and GBM1063 in a 4.2 cM interval. EST-derived microsatellite markers were used to establish the syntenic relationships to the genomic rice sequences. Two orthologous sites on rice chromosome 2 flanking a 4.1 Mb sequence had homology to the respective barley markers in the sex1 region. For the markers in the seg8 region orthologous sites on rice chromosome 6 were detected.

Chromosome Mapping↗

REGULATION OF FATTY ACID SYNTHESIS.

All plant cells produce fatty acids from acetyl-CoA by a common pathway localized in plastids. Although the biochemistry of this pathway is now well understood, much less is known about how plants control the very different amounts and types of lipids produced in different tissues. Thus, a central challenge for plant lipid research is to provide a molecular understanding of how plants regulate the major differences in lipid metabolism found, for example, in mesophyll, epidermal, or developing seed cells. Acetyl-CoA carboxylase (ACCase) is one control point that regulates rates of fatty acid synthesis. However, the biochemical modulators that act on ACCase and the factors that in turn control these modulators are poorly understood. In addition, little is known about how the expression of genes involved in fatty acid synthesis is controlled. This review evaluates current knowledge of regulation of plant fatty metabolism and attempts to identify the major unanswered questions.

Journal Article↗

PHLOEM UNLOADING: Sieve Element Unloading and Post-Sieve Element Transport.

The transport events from the sieve elements to the sites of utilization within the recipient sink cells contribute to phloem unloading. The phenomenon links sink metabolism and/or compartmentation with phloem transport to, and partitioning between, sinks. The nature of the linkage depends upon the cellular pathway and mechanism of unloading. The common unloading pathway is symplasmic, with an apoplasmic step at or beyond the sieve element boundary reserved for specialized situations. Plasmodesmal conductivity exerts the primary control over symplasmic transport that occurs by diffusion with bulk flow anticipated to be of increasing significance as import rate rises. In the case of an apoplasmic step, efflux across the plasma membranes of the vascular cells occurs by simple diffusion, whereas efflux from nonvascular cells of developing seeds is facilitated and, in some cases, energy coupled. Accumulation of sugars from the sink apoplasm universally occurs by a plasma membrane-bound sugar/proton symport mechanism.

Journal Article↗

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability.

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.

Cloning, Molecular↗

Distribution of gibberellins and expressional analysis of GA 20-oxidase genes of morning glory during fruit maturation.

Gibberellins A1/3 (GA1/3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1/3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1/3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible alpha-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.

Fruit↗

Genomic imprinting in plants.

Genomic imprinting, though most extensively studied in mammals, has long been known to perform an important role in seed development in flowering plants. In this chapter, an overview of what is known to date about genomic imprinting in flowering plants and how this knowledge came into being will be given.

Genomic Imprinting↗

Action windows for optimum control of rangeland grasshoppers (Orthoptera: Acrididae).

Different assemblages of primary and secondary pest grasshopper species were found in 5 different rangeland plant communities in western North Dakota. The action window for their control (i.e., the interval when control is likely to be efficacious) with short-lived insecticides can be visualized in terms of time, grasshopper phenology, or plant phenology. Action windows ranged from 14 d duration in habitats dominated by western wheatgrass to 38 d duration in habitats dominated by needle-and-thread or by crested wheatgrass. Action windows opened 11-15 d later in rhizominous grass habitats than in bunchgrass habitats because of increased presence of late-hatching secondary pest species. In all habitat types, action windows closed primarily in response to imminent oviposition by a ubiquitous primary pest species, Melanoplus sanguinipes (F.). In all habitat types, action windows tended to open during seed development of 2 common and conspicuous grasses, needle-and-thread and green needlegrass. No such consistent indicator for closure of action windows was detected.

Animals↗

An opaque-2-like transcription factor from pearl millet.

Pearl millet (Pennisetum glaucum L.), a species of the Poaceae family, is an important food crop in Africa, Asia and South America. Its nutritional value is due to storage prolamins accumulated in the seeds. In other species of the same family, the expression of the genes coding for storage prolamins is mediated by the regulatory protein opaque-2. In this paper we show that an opaque-2 -like protein is present in pearl millet too and is expressed during the early stages of seed development. The organization of the gene coding for this protein is similar to that of orthologous genes in other Poaceae species, i.e. six exons separated by five introns. A comparison of amino acid homologies with other described opaque-2 proteins is presented.

Amino Acid Sequence↗

Differential expression of the Arabidopsis genes coding for Em-like proteins.

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).

Amino Acid Motifs↗

The role of fructan in flowering of Campanula rapunculoides.

Inulin type fructan was detected in all vegetative organs of Campanula rapunculoides L. plants. All flower parts contained fructan at some developmental stage. A steady decrease was found in sepals during development. Petals, however, stored fructan in the bud stage. A rapid breakdown during opening of the flower resulted in high concentrations of glucose and especially fructose that may contribute to the osmotic driving force involved in petal expansion. Before complete shrivelling, the hexoses were apparently exported from flower parts. Fructans were hydrolysed and exported from the stamen and style tissue upon flower opening. Similarly, the major fructan reserves in the ovary were broken down almost simultaneously with those in other flower parts. Hexoses did not reach high levels in the ovary, probably because they were rapidly metabolized and/or incorporated by developing seeds.

Chromatography, Ion Exchange↗

Does triacylglycerol biosynthesis require diacylglycerol acyltransferase (DAGAT)?

Microsomal membrane preparations from the developing seeds of sunflower (Helianthus annuus L.) catalyse the conversion of sn-glycerol-3-phosphate and acyl-CoA to triacylglycerol via phosphatidic acid and diacylglycerol. The formation of diacylglycerol from phosphatidic acid was Mg2+ dependent and in the presence of EDTA phosphatidic acid accumulated. This property was used to generate large quantities of endogenous radioactive phosphatidic acid in the membranes. On addition of Mg2+ the phosphatidic acid was used in triacylglycerol formation. Acyl-CoA had little effect on the label which accumulated in triacylglycerol from phosphatidic acid. Diacylglycerol acyltransferase, therefore, may not play a major role in oil formation as originally envisaged and other enzymes, including diacylglycerol:diacylglycerol transacylase [Stobart, Mancha, Lenman, Dahlqvist and Stymne (1997) Planta 203, 58-66] may have important biosynthetic functions.

Acyl Coenzyme A↗

Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea.

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea, genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in lambdaEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone lambda5.12 after a series of screening, sub-cloning and Southern hybridization.

Brassica↗

Investigating the hows and whys of DNA endoreduplication.

Endoreduplication is a form of nuclear polyploidization that results in multiple, uniform copies of chromosomes. This process is common in plants and animals, especially in tissues with high metabolic activity, and it generally occurs in cells that are terminally differentiated. In plants, endoreduplication is well documented in the endosperm and cotyledons of developing seeds, but it also occurs in many tissues throughout the plant. It is thought that endoreduplication provides a mechanism to increase the level of gene expression, but the function of this process has not been thoroughly investigated. Numerous observations have been made of endoreduplication, or at least extra cycles of S-phase, as a consequence of mutations in genes controlling several aspects of cell cycle regulation. However, until recently there were few studies directed at the molecular mechanisms responsible for this specialized cell cycle. It is suggested that endoreduplication requires nothing more elaborate than a loss of M-phase cyclin-dependent kinase activity and oscillations in the activity of S-phase cyclin-dependent kinase.

Cell Cycle↗

The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture.

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.

Arabidopsis↗

Travelling seeds? Examples from New Zealand.

New Zealand is an archipelago isolated in time and space. Flowering plants have evolved in the almost-complete absence of terrestrial mammals, some of the most prolific seed-dispersers from the rest of the world, and fascinating mechanisms for spreading seeds have developed.

Animals↗