The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.     

C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes

A carrot gene homologous to the ABI3 gene of Arabidopsis was isolated from a carrot somatic embryo cDNA library and designated C-ABI3. The sequence of C-ABI3 was very similar to those of ABI3 of Arabidopsis and VP1 of maize in certain conserved regions. The expression of C-ABI3 was detected specifically in embryogenic cells, somatic embryos and developing seeds. Thus, expression of C-ABI3 was limited to tissues that acquired desiccation tolerance in response to endogenous or exogenous abscisic acid (ABA). Endogenous levels of ABA in seeds increased transiently and then desiccation of seeds started. The expression of C-ABI3 in developing seeds was observed prior to the increase in levels of endogenous ABA that was followed by desiccation of seeds. In transgenic mature leaves in which C-ABI3 was ectopically expressed, expression of ECP31, ECP63 and ECP40 was induced by treatment with ABA, which indicates that the expression of ECP genes was controlled by the pathway(s) that involved C-ABI3 and ABA. This suggests that C-ABI3 has the same function as VP1/ABI3 factor in carrot somatic embryos.     

Rice embryo globulins: amino-terminal amino acid sequences, cDNA cloning and expression

A globulin fraction prepared from rice embryos contained polypeptides or polypeptide groups of 49 kDa (designated REG1), 46 kDa (designated REG2), about 35 kDa, 32 kDa and 25 kDa. The amino-terminal sequences of REG1 and the major polypeptide in the 35-kDa group were identical, suggesting that the REG1 polypeptide undergoes partial proteolytic processing that removes a carboxy-terminal region. A cDNA clone, designated pcREG2, encoding REG2 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence of REG2 was found to be 68% identical to that of the maize GLB2 globulin. Reg2 mRNA was present at high levels during embryo development for up to 14 days after flowering (DAF). Lower levels were found 20 DAF when the maturation of embryos was almost completed, and at the dry mature stage. Reg2 mRNA almost disappeared upon imbibition of isolated dry mature embryos but it was re-induced at a low level by further treatment with ABA. The expression of Reg2 was not induced by ABA in suspension-cultured cells, unlike that of Osem, one of the late embryogenesis abundant protein (LEA) genes.     

Phenotypic analysis and molecular cloning of discolored-1 (dsc1), a maize gene required for early kernel development

Recessive mutations in the maize dsc1 locus prevent normal kernel development. Solidification of the endosperm in homozygous dsc1- mutant kernels was undetectable 12 days after pollination, at which time the tissue was apparently completely solidified in wild-type kernels. At later times endosperm did solidify in homozygous dsc1- mutant kernels, but there was a marked reduction in the volume of the tissue. Embryo growth in homozygous dsc1- kernels was delayed compared to wild-type kernels, but proceeded to an apparently normal stage 1 in which the scutellum, coleoptile, and shoot apex were clearly defined. Embryo growth then ceased and the embryonic tissues degraded. Late in kernel development no tissue distinctions were obvious in dsc1- mutant embryos. Immature mutant embryos germinated when transplanted from kernels to tissue culture medium prior to embryonic degeneration, but only coleoptile proliferation was observed. The dsc1 gene was isolated by transposon tagging. Analysis of the two different dsc1- mutations confirmed that transposon insertion into the cloned genomic locus was responsible for the observed phenotype. Dsc1 mRNA was detected specifically in kernels 5-7 days after pollination. These data indicate Dsc1 function is required for progression of embryo development beyond a specific stage, and also is required for endosperm development.     

mex-1 and the general partitioning of cell fate in the early C. elegans embryo

It is thought that at least some of the initial specification of the five somatic founder cells of the C. elegans embryo occurs cell-autonomously through the segregation of factors during cell divisions. It has been suggested that in embryos from mothers homozygous for mutations in the maternal-effect gene mex-1, four blastomeres of the 8-cell embryo adopt the fate of the MS blastomere. It was proposed that mex-1 functions to localise or regulate factors that determine the fate of this blastomere. Here, a detailed cell lineage analysis of 9 mex-1 mutants reveals that the fates of all somatic founder cells are affected by mutations in this gene. We propose that mex-1, like the par genes, is involved in establishing the initial polarity of the embryo.     

Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis

Hox genes are expressed in dynamic patterns during embryogenesis consistent with their role in axial specifications. To study the distribution of mouse Hoxc8, a homeodomain containing protein, we raised monoclonal antibodies against the least conserved portion of Hoxc8. Using these antibodies, we have examined early and mid-gestation embryos for the distribution of the protein. At the end of gastrulation Hoxc8 is expressed in the caudal portion of the embryo. In the neural tube, an early phase when all cells express Hoxc8 is distinguished from a late phase with predominant expression in differentiating neurons. A comparison of this expression pattern with that of a reporter gene under the control of the early Hoxc8 enhancer demarcates three separate regulatory components: (1) initiation and establishment; (2) maintenance; and (3) downregulation. We propose that Hoxc8 expression during embryogenesis is established in multiple phases. Possible regulatory mechanisms involved in generating such a complex domain of Hox gene expression are discussed.     

Effects of carbohydrate and lipid on resting energy expenditure, heart rate, sleepiness, and mood

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

Molecular analysis of protein domain function encoded by the myb-homologous maize genes C1, Zm 1 and Zm 38

Two maize genes, Zm 1 and Zm 38, related to the regulatory anthocyanin gene C1 were analyzed molecularly and used for fusion constructs in transient domain swapping experiments with the C1 wild-type gene. It was shown that both genes (Zm 1 and Zm 38) influence the expression of the A1 locus, a target gene for C1. Zm 1 activates the A1 promoter, however it does not turn on the whole anthocyanin pathway. The Zm 38 gene product shows functions similar to C1-I, a dominant inhibitor of the C1 wild-type gene. Concerning the trans-inhibition by C1-I two effects seem to be involved, competition for binding and formation of heterodimers. Further analysis of C1 function was carried out by a fine structure analysis of C1 mutants induced by the insertion and excision of transposable elements. These experiments indicate that for the activating domain of the protein, the formation of an alpha helix seems to be more important than a high negative charge.     

Genetic regulation of preimplantation mouse embryo survival

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as alpha-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium

Inducible virulence (vir) genes of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid are under control of a two-component regulatory system. In response to environmental factors (phenolic compounds, sugars, pH) VirA protein phosphorylates VirG, which in turn interacts with the promoters of other vir genes, causing induction. A mutation of virG, virGN54D (which codes for a Asn-54-->Asp amino acid change in the product), causes constitutive expression of other vir genes independent of virA. We have investigated whether providing Agrobacterium with a plasmid containing virGN54D augments the efficiency of transfer of the T-DNA (transferred DNA). For both tobacco and cotton, we observed an enhancement of transformation efficiency when the inciting Agrobacterium strain carries the virGN54D mutation. We also tested whether supplying Agrobacterium with a similar plasmid containing wild-type virG affects the efficiency of T-DNA transfer. An intermediate efficiency was observed when this plasmid was employed. Using a beta-glucuronidase (GUS) reporter gene to assess transient expression of T-DNA after transfer to tobacco and maize tissues, we observed a higher frequency of GUS-expressing foci after inoculation with Agrobacterium strains carrying virGN54D than with Agrobacterium carrying the wild-type virG. Gene-transfer efficiency to maize by an octopine strain was greatly improved upon introduction of virGN54D. Multiple copies of wild-type virG were equally effective in promoting transient expression efficiency in tobacco but were virtually ineffective in maize. We propose the use of virGN54D to improve the efficiency of Agrobacterium-mediated transformation, especially for recalcitrant plant species.