A Dual-Promoter Gene Orchestrates the Sucrose-Coordinated Synthesis of Starch and Fructan in Barley.

Sequential carbohydrate synthesis is important for plant survival because it guarantees energy supplies for growth and development during plant ontogeny and reproduction. Starch and fructan are two important carbohydrates in many flowering plants and in human diets. Understanding this coordinated starch and fructan synthesis and unraveling how plants allocate photosynthates and prioritize different carbohydrate synthesis for survival could lead to improvements to cereals in agriculture for the purposes of greater food security and production quality. Here, we report a system from a single gene in barley employing two alternative promoters, one intronic/exonic, to generate two sequence-overlapping but functionally opposing transcription factors, in sensing sucrose, potentially via sucrose/glucose/fructose/trehalose 6-phosphate signaling. The system employs an autoregulatory mechanism in perceiving a sucrose-controlled trans activity on one promoter and orchestrating the coordinated starch and fructan synthesis by competitive transcription factor binding on the other promoter. As a case in point for the physiological roles of the system, we have demonstrated that this multitasking system can be exploited in breeding barley with tailored amounts of fructan to produce healthy food ingredients. The identification of an intron/exon-spanning promoter in a hosting gene, resulting in proteins with distinct functions, adds to the complexity of plant genomes.

Tropomyosin is a tetramer under physiological salt conditions.

Tropomyosin (TM) is a coiled-coil dimer of alpha-helical peptides, which self associates in a head- to-tail fashion along actin polymers, conferring stability to the microfilaments and serving a regulatory function in acto-myosin driven force generation. While the major amount of TM is associated with filaments also in non-muscle cells, it was recently reported that there are isoform-specific pools of TM multimers (not associated with F-actin), which appear to be utilized during actin polymerization and reformed during depolymerization. To determine the size of these multimers, skeletal muscle TM was studied under different salt conditions using gel-filtration and sucrose gradient sedimentation, and compared with purified non-muscle TM 1 and 5, as well as with TM present in non-muscle cell extracts and skeletal muscle TM added to such extracts. Under physiological salt conditions TM appears as a single homogenous peak with the Stokes radius 8.2 nm and the molecular weight (mw) 130,000. The corresponding values for TM 5 are 7.7 nm and 104,000, respectively. This equals four peptides, implying that native TM is a tetramer in physiological salt. It is therefore concluded that the TM multimers are tetramers.

Myonuclear domain size and myosin isoform expression in muscle fibres from mammals representing a 100,000-fold difference in body size.

This comparative study of myonuclear domain (MND) size in mammalian species representing a 100,000-fold difference in body mass, ranging from 25 g to 2500 kg, was undertaken to improve our understanding of myonuclear organization in skeletal muscle fibres. Myonuclear domain size was calculated from three-dimensional reconstructions in a total of 235 single muscle fibre segments at a fixed sarcomere length. Irrespective of species, the largest MND size was observed in muscle fibres expressing fast myosin heavy chain (MyHC) isoforms, but in the two smallest mammalian species studied (mouse and rat), MND size was not larger in the fast-twitch fibres expressing the IIA MyHC isofom than in the slow-twitch type I fibres. In the larger mammals, the type I fibres always had the smallest average MND size, but contrary to mouse and rat muscles, type IIA fibres had lower mitochondrial enzyme activities than type I fibres. Myonuclear domain size was highly dependent on body mass in the two muscle fibre types expressed in all species, i.e. types I and IIA. Myonuclear domain size increased in muscle fibres expressing both the beta/slow (type I; r = 0.84, P < 0.001) and the fast IIA MyHC isoform (r = 0.90; P < 0.001). Thus, MND size scales with body size and is highly dependent on muscle fibre type, independent of species. However, myosin isoform expression is not the sole protein determining MND size, and other protein systems, such as mitochondrial proteins, may be equally or more important determinants of MND size.

Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology.

Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells.

The microfilament system and malignancy.

Increased motile activity, increased rate of cell proliferation and removal of growth inhibiting cell-cell contacts are hallmarks of tumorigenesis. Activation of cell motility and migration is caused by activation of receptors, turning on the growth cycle. Increased expression of metalloproteinases, breaking cell:cell contacts and organ confines, allows the spread of malignant cancer cells to other sites in the organism. It has become increasingly clear that most transmembrane proteins (growth factor receptors, adhesion proteins and ion channels) are either permanently or transiently associated with the sub-membraneous system of actin microfilaments (MF), whose force generating capacity they control. Although there has been great progress in our understanding of the physiological importance of the MF-system, as will be exemplified in this issue of SCB, many aspects of actin microfilament formation and its regulation are still unclear. Redox control of the actin (MF)-system in cell motility and migration and its perturbations in pathophysiology, including cancer, is an emerging field of research.

Sweet delivery - sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells.

Antisense oligodeoxynucleotides (ODNs) are short (12-25 nt long) stretches of single-stranded DNA that may be delivered to a cell, where they hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. Here we used confocal microscopy to monitor the uptake and trafficking of ODNs in barley tissues. We conclude that uptake of ODNs across the plant plasma membrane is mediated by active transport of mono- or disaccharides through sugar translocators. We demonstrate that sugar transport can deliver ODNs to barley seeds, and that this strategy may be employed to suppress gene activity in endosperm cells by antisense ODN inhibition. We further found that sucrose compared favorably with oligofectamine as a vehicle for ODN delivery to human cells in a low-serum environment.

Transcription factors in muscle atrophy caused by blocked neuromuscular transmission and muscle unloading in rats.

The muscle wasting associated with long-term intensive care unit (ICU) treatment has a negative effect on muscle function resulting in prolonged periods of rehabilitation and a decreased quality of life. To identify mechanisms behind this form of muscle wasting, we have used a rat model designed to mimic the conditions in an ICU. Rats were pharmacologically paralyzed with a postsynaptic blocker of neuromuscular transmission, and mechanically ventilated for one to two weeks, thereby unloading the limb muscles. Transcription factors were analyzed for cellular localization and nuclear concentration in the fast-twitch muscle extensor digitorum longus (EDL) and in the slow-twitch soleus. Significant muscle wasting and upregulation of mRNA for the ubiquitin ligases MAFbx and MuRF1 followed the treatment. The IkappaB family-member Bcl-3 displayed a concomitant decrease in concentration, suggesting altered kappaB controlled gene expression, although NFkappaB p65 was not significantly affected. The nuclear levels of the glucocorticoid receptor (GR) and the thyroid receptor alpha1 (TRalpha1) were altered and also suggested as potential mediators of the MAFbx- and MuRF1-induction in the absence of induced Foxo1. We believe that this model, and the strategy of quantifying nuclear proteins, will provide a valuable tool for further, more detailed, analyses of the muscle wasting occurring in patients kept on a mechanical ventilator.

Developmental regulation of a VEIDase caspase-like proteolytic activity in barley caryopsis.

Caspases are essential in animal programmed cell death both as initiator and executioner proteases. Plants do not have close caspase homologues, but several instances of caspase-like proteolytic activity have been demonstrated in connection with programmed cell death in plants. It was asked if caspase-like proteases are involved during development of the barley caryopsis. The presence of a caspase-6-like proteolytic activity that preferentially cleaved the sequence VEID was demonstrated. A range of protease inhibitors was tested and only caspase-specific inhibitors showed major inhibitory effects. The profile of VEIDase activity in developing starchy endosperm, embryo, and whole caryopsis was measured and showed a general trend of higher activity in young, rapidly developing tissues. The VEIDase activity was localized in vivo to vesicles, shown to be autophagosomes, in randomly distributed cells of the starchy endosperm. The VEIDase activity detected in barley caryopsis is similar to activities described previously in mammals, spruce, yeast, and thale cress. In mammals, spruce, and yeast, VEIDase activity has been shown to be positively correlated with the occurrence of programmed cell death. Several manifestations of programmed cell death exist in developing barley caryopsis, indicating a connection between VEIDase activity and developmental programmed cell death in barley.

Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling.

Sugar signalling cascades are important components of regulatory networks in cells. Compared with the situation in bacteria, yeast and animals, participants of the sugar signalling pathways in plants are poorly understood. Several genes involved in starch synthesis are known to be sugar inducible, although the signal transduction pathways remain undisclosed. We reported recently the isolation of SUSIBA2, a transcription factor involved in sugar-mediated regulation of starch synthesis. Here, we used antisense oligodeoxynucleotide (ODN) inhibition, a powerful approach in medical sciences, to block the effects of SUSIBA2 in sugar-treated barley leaves. The uptake and intracellular trafficking of an 18-mer susiba2 antisense ODN in leaves were followed by confocal microscopy. Administration of the antisense ODN to the leaves impeded susiba2 expression by RNase H activation. This dramatically diminished the ectopic expression of the iso1 and sbeIIb genes and resulted in altered starch synthesis. This study illustrates the successful exploitation of the antisense ODN technology in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and identifies SUSIBA2 as a transcriptional activator in plant sugar signalling. Based on our findings, we propose a model for sugar-signalling control of starch synthesis.

Synthesis of ketocarotenoids in the seed of Arabidopsis thaliana.

A cDNA coding for a gene necessary for synthesis of ketocarotenoids was cloned from the alga Haematococcus pluvialis and expressed in the seed of Arabidopsis thaliana. The expression of the algal beta-carotene-oxygenase gene was directed to the seed by use of the 2S, seed storage protein promoter napA. Extracts from seeds of the transgenic plants were clearly red because of accumulation of ketocarotenoids, and free and esterified forms of ketocarotenoids were found in addition to the normal carotenoid composition in the seed. The major ketocarotenoids in the transgenic plants were: 4-keto-lutein (3,3'-dihydroxy-beta-,epsilon-carotene-4-one), adonirubin (3-hydroxy-beta-,beta'-carotene-4,4'-dione) and canthaxanthin (beta-,beta'-carotene-4,4'-dione). 4-Keto-lutein differs from the more common adonixanthin only in the position of one double bond. To increase the substrate availability for the beta-carotene-oxygenase, these transformants were crossed with transgenic plants overexpressing a construct of an endogenous phytoene synthase gene, also under the control of the napA promoter. The resulting crossings gave rise to seeds with a 4.6-fold relative increase of the total pigment, and the three major ketocarotenoids were increased 13-fold compared to seeds of transgenic plants carrying only the beta-carotene-oxygenase construct.

Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid.

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of beta-carotene and a total average amount of beta-carotene of approximately 260 microg g-1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and alpha-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.

Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins.

Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

An antigen expressed during plant vascular development crossreacts with antibodies towards KLH (keyhole limpet hemocyanin).

An antigen present in plant vascular tissue crossreacts with antibodies towards keyhole limpet hemocyanin (KLH). The antigen is present in xylem and vascular cambium, as evidenced by immunocytochemical staining of plant sections. This cell type assignment was confirmed by staining of mesophyll cell cultures from Zinnia elegans L. undergoing tracheary cell differentiation. The strongest staining both in sections and cell cultures occurred in cells and tissues during early stages of differentiation. Although the anti-KLH antibodies can easily be removed by affinity purification, our findings suggest that a certain caution is needed when KLH is used as an immunological carrier for studies in plants.