Abstract: Mutation of the core pentamer, CCGAC, of two putative low temperature responsive elements (LTREs) in the 5'-proximal region for the winter Brassica napus cold-induced gene BN115 was carried out. Analyses of transient expression of the resultant mutated BN115 promoter-GUS fusions revealed the loss of low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation of two G-boxes, CACGTG, staggered between the LTREs in the same region of the promoter did not alter cold-inducible gene expression. Replacement of a possible enhancer region of the BN115 promoter with the enhancer from the CaMV 35S promoter resulted in a several-fold increase in low temperature-induced GUS activity.

Abstract: The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here.

Abstract: We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5′-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl2 which delayed loss of chlorophyll but not that of photochemical efficiency.

Abstract: The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to theuidA reporter gene to test promoter expression. The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene. Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions. The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants. The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression. In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips.

Abstract: In the mitochondria and chloroplasts of higher plants there is an RNA editing activity responsible for specific C-to-U conversions and for a few U-to-C conversions leading to RNA sequences different from the corresponding DNA sequences. RNA editing is a post-transcriptional process which essentially affects the transcripts of protein coding genes, but has also been found to modify non-coding transcribed regions, structural RNAs and intron sequences. RNA editing is essential for correct gene expression: proteins translated from edited transcripts are different from the ones deduced from the genes sequences and usually present higher similarity to the corresponding non-plant homologues. Initiation and stop codons can also be created by RNA editing. RNA editing has also been shown to be required for the stabilization of the secondary structure of introns and tRNAs. The biochemistry of RNA editing in plant organelles is still largely unknown. In mitochondria, recent experiments indicate that RNA editing may be a deamination process. A plastid transformation technique showed to be a powerful tool for the study of RNA editing. The biochemistry as well as the evolutionary features of RNA editing in both organelles are compared in order to identify common as well as organelle-specific components.

Abstract: The biosynthesis of the photosynthetic apparatus depends on the concerted action of the nuclear and chloroplast enetic systems. Numerous nuclear and chloroplast mutants of Chlamydomonas deficient in photosynthetic activity have been isolated and characterized. While several of these mutations alter the genes of components of the photosynthetic complexes, a large number of the mutations affect the expression of chloroplast genes involved in photosynthesis. Most of these mutations are nuclear and only affect the expression of a single chloroplast gene. The mutations examined appear to act principally at post-transcriptional steps such as RNA stability, RNA processing, cis- and trans-splicing and translation. Directed chloroplast DNA surgery through biolistic transformation has provided a powerful tool for identifying important cis elements involved in chloroplast gene expression. Insertion of chimeric genes consisting of chloroplast regulatory regions fused to reporter genes into the chloroplast genome has led to the identification of target sites of the nuclear-encoded functions affected in some of the mutants. Biochemical studies have identified a set of RNA-binding proteins that interact with the 5′-untranslated regions of plastid mRNAs. The binding activity of some of these factors appears to be modulated by light and by the growth conditions.

Abstract: 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.

Abstract: Blue-light responses are evolutionarily among the most ancient and diverse of light-regulatory phenomena. Many such responses in algal, bacterial, or fungal systems show action spectra that are consistent with a flavin chromophore, with peaks of activity in the near-UV (around 350 nm) and blue (450-480 nm) regions of the spectrum. In higher plants, specific blue-light responses have been documented for over a century [20, 68], and include phototropism, inhibition of hypocotyl elongation, stomatal opening, anthocyanin production, and expression of specific blue lightregulated genes. Action spectra compiled from a number of such responses fit the general criteria of a flavin-type blue light photoreceptor; however, due in part to the difficulty of obtaining a good in vitro assay system for flavin-type blue light regulated phenomena, and in part to the numerous non-photoreceptor pigments absorbing in the blue (for instance, cytochromes, chlorophylls, carotenoids, and flavins bound to metabolic enzymes), there has until very recently been little progress towards identification of a receptor. In keeping with their elusive chemical identity, the name 'cryptochrome' has been proposed for such B-type receptors [26]. It would be impossible in the space of such a short review to do justice to the immense literature that has accumulated on blue light responses and signalling pathways even within the past few years. We have chosen therefore to focus primarily on a major milestone in the field: the recent first isolation of a blue-light photoreceptor, CRY1 (for cryptochrome), from Arabidopsis thaliana by molecular genetic techniques. Additional developments in blue light perception, which have been addressed extensively in a number of excellent recent reviews and monographs [5, 9, 13, 14, 21, 23, 24, 28, 29, 33, 34, 35, 44, 53, 60, 72, 73, 79, 80] will be covered here primarily as they relate to the newly identified CRY 1 photoreceptor.

Abstract: Our understanding of how the 3′ ends of mRNAs are formed in plants is rudimentary compared to what we know about this process in other eukaryotes. The salient features of plant pre-mRNAs that signal cleavage and polyadenylation remain obscure, and the biochemical mechanism is as yet wholly uncharacterised. Nevertheless, despite the lack of universally conserved cis-acting motifs, a common underlying architecture is emerging from functional analyses of plant poly(A) signals, allowing meaningful comparison with components of poly(A) signals in other eukaryotes. A plant poly(A) signal consists of one or more near-upstream elements (NUE), each directing processing at a poly(A) site a short distance downstream of it, and an extensive far-upstream element (FUE) that enhances processing efficiency at all sites. By analogy with other systems, a model for a plant 3′-end processing complex can be proposed. Plant poly(A) polymerases have been isolated and partially characterised. These, together with hints that some processing factors are conserved in different organisms, opens promising avenues toward initial characterisation of the trans-acting factors involved in 3′-end formation of mRNAs in higher plants.

Abstract: This review examines the biogenesis of the plant storage lipid organelle known as the oil body. Although oil body is the most commonly used for this organelle, it is sometimes referred to as the oleosome or spherosome. The role of the oil body is accepted as being a subcellular compartment in which lipid reserves are deposited and stored. These reserves, in the form of triacytgycerols (TAG), make up the bulk of the oil body. The TAG is then broken down and metabolized in energy-consumptive events such as germination (see [16, 21,42]) for recent reviews). Oil bodies are found in abundance only in oil-storing tissues where reserve mobilization occurs, such as seeds and pollen, but appear not to be present in off-containing tissues such as olive drupes (in which the storage lipid is not used for germination) where the storage lipid is coalesced into large undefined droplets [42]. It is generally concluded, therefore, that the oil body may facilitate the efficient breakdown of the storage lipid because of its high surface area to volume ratio (i.e. a greater area of TAG available for breakdown) and thus allow more efficient catabolism. Certainly the oil bodies in seeds of a wide range of different plant species appear to be of a similar size, usually ranging between 0.2 and 21*m in diameter [58], though these estimations are based on electron microscopy of ultrathin sections of seed tissue, whereas the oil bodies are usually highly compressed in the cell in vivo. In addition, the size of the oil body may increase over the course of seed development and maturation, as do the number of oil bodies, until they are the predominant organelle in the oil-storing tissues, occupying the cytoplasm of the cell [9]. The structure of the oil body appears to be simple with the storage TAG in the lumen making up the vast majority of its volume. However, a discrete boundary layer is present on the outer, cytosolic, face of the oil body. This was initially described as a pellicle and examination under the electron microscope (EM) gave measurements of 2-4 nm in thickness. This thickness is less than that expected of a normal (unit) membrane, as found defining other organelle, marking the oil body as being unusual. Moreover, if the oil bodies are washed with solvents such as diethyl ether to extract the TAG without disrupting the pellicle, then the collapsed structures (\"ghosts\") observed by EM look similar to a unit membrane. This has led to the hypothesis that the oil body may be bounded by a half-unit membrane [21,23].

Abstract: The molecular mechanisms that initiate and control the metabolic activities of seed germination are largely unknown. Sugars may play important roles in regulating such metabolic activities in addition to providing an essential carbon source for the growth of young seedlings and maintaining turgor pressure for the expansion of tissues during germination. To test this hypothesis, we investigated the physiological role of sugars in the regulation of alpha-amylase gene expression and carbohydrate metabolism in embryo and endosperm of germinating rice seeds. RNA gel blot analysis revealed that in the embryo and aleurone cells, expression of four alpha-amylase genes was differentially regulated by sugars via mechanisms beyond the well-known hormonal control mechanism. In the aleurone cells, expression of these alpha-amylase genes was regulated by gibberellins produced in the embryo and by osmotically active sugars. In the embryo, expression of two alpha-amylase genes and production of gibberellins were transient, and were probably induced by depletion of sugars in the embryo upon imbibition, and suppressed by sugars influx from the endosperm as germination proceeded. The deferential expression of the four alpha-amylase genes in the embryo and aleurone cells was probably due to their markedly different sensitivities to changes in tissue sugar levels. Our study supports a model in which sugars regulate the expression of alpha-amylase genes in a tissue-specific manner: via a feedback control mechanism in the embryo and via an osmotic control mechanism in the aleurone cells. An interactive loop among sugars, gibberellins, and alpha-amylase genes in the germinating cereal grain is proposed.

Abstract: Dominant mutations in the Arabidopsis ETR1 gene block the ethylene signal transduction pathway. The ETR1 gene has been cloned and sequenced. Using the ETR1 cDNA as a probe, we identified a cDNA homologue (eTAE1) from tomato. eTAE1 contains an open reading frame encoding a polypeptide of 754 amino acid residues. The nucleic acid sequence for the coding sequence in eTAE1 is 74% identical to that for ETR1, and the deduced amino acid sequence is 81% identical and 90% similar. Genomic Southern blot analysis indicates that three or more ETR1 homologues exist in tomato. RNA blots show that eTAE1 mRNA is constitutively expressed in all the tissues examined, and its accumulation in leaf abscission zones was unaffected by ethylene, silver ions (an inhibitor of ethylene action) or auxin.

Abstract: The terminal process of xylogenesis, autolysis, is essential for the formation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H2O2 challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.

Abstract: The barley BARE-1 is a transcribed, copia-like retroelement with well-conserved functional domains, an active promoter, and a copy number of at least 3 × 104. We examined its chromosomal localization by in situ hybridization. The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5′-untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a targe site preference.

Abstract: This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.

Abstract: The complexity associated with post-translational processing, in terms of protein sorting and delivery is now well understood. Although such studies have been focused almost exclusively on the fate of proteins within the cell in which they are synthesized, recent studies indicate that it is time to broaden this focus to incorporate the concept of intercellular targeting of proteins. Direct evidence is now available that viral and endogenous proteins can be synthesized in a particular cell and subsequently transported into neighboring (or more distant) cells. Plasmodesmata, plasma membrane-lined cytoplasmic pores, are thought to establish the intercellular pathway responsible for this cell-to-cell trafficking of macromolecules (proteins and nucleic acids). These recent findings establish a new paradigm for understanding the manner in which higher plants exert control over developmental processes. We discuss the concept that programming of plant development involves supracellular control achieved by plasmodesmal trafficking of informational molecules, herein defined as supracellular control proteins (SCPs). This novel concept may explain why, in plants, cell fate is determined by position rather than cell lineage. Finally, the circulation of long-distance SCPs, within the phloem, may provide the mechanism by which the plant signals to the shoot apical meristem that it is time to switch to the reproductive phase of its development.

Abstract: The molecular mechanism of signal transduction pathways which mediate the action of phytohormones are poorly understood. Recently, we and others have shown that the as -1 type cis-acting elements can respond to auxin and salicylic acid, two well-characterized signaling molecules in plants. In the present work, we have examined a comprehensive set of physiological and abiotic agents and found that auxin, salicylic acid and methyl-jasmonate are three effective inducers of the as-1-type elements in transgenic tobacco. Using a cell suspension culture containing a synthetic promoter-GUS fusion, we demonstrated rapid and sensitive induction of the as-1-type element by these phytohormones. Furthermore, a tobacco glutathione S-transferase gene, GNT35, that contains an as-1-type binding site in its promoter is also inducible by auxin, salicylic acid and methyl-jasmonate with similar kinetics. As Ulmasov et al. have recently reported, we found that the as-1-type elements can also respond to weak/inactive analogues of auxin and salicylic acid. In addition, we show that hydrogen peroxide can also effectively activate the expression of GNT35 as well as the as-1-type element in a cell suspension culture, but not with whole seedlings. These results are discussed with respect to the possible mechanism(s) through which a single cis element may respond to a diverse array of molecules.

Abstract: The BARE-1 retrotransposon occurs in more than 10(4) copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.

Abstract: We have analysed 998 Arabidopsis intron sequences in the EMBL database. All Arabidopsis introns to adhere to the :GU...AG: rule with the exception of 1% of introns with :GC at their 5′ ends. Virtually all of the introns contained a putative branchpoint sequence (YUNAN) 18 to 60 nt upstream of the 3′ splice site. Although a polypyrimidine tract was much less apparent than in vertebrate introns, the most common nucleotide in the region upstream of the 3′ splice site was uridine. Consensus sequences for 5′ and 3′ splice sites and branchpoint sequences for Arabidopsis introns are presented.

Abstract: To study regulation of the plastid-localized maize carotenoid biosynthetic pathway, a cDNA encoding phytoene desaturase (PDS) was isolated and characterized. The DNA sequence of the maize Pds cDNA was determined and compared with available dicot Pds genes. The deduced PDS protein, estimated at 64.1 kDa (unprocessed), had a dinucleotide binding domain and conserved regions characteristic of other carotene desaturases. Alignment of available PDS sequences from distantly related organisms suggests that Pds has potential as a phylogenetic tool. By use of heterologous complementation in Escherichia coli, maize PDS was shown to catalyze two desaturation steps converting phytoene to ζ-carotene. RFLP (restriction fragment length polymorphism) mapping was used to place Pds on chromosome 1S near viviparous5 (vp5), and RT-PCR (reverse-transcriptase polymerase chain reaction) analysis indicated reduced Pds transcript in vp5 mutant relative to normal endosperm. Other phytoene-accumulating mutant endosperms, vp2 and white3 (w3), showed no difference in Pds transcript accumulation as compared with normal endosperm counterparts. RT-PCR analysis of Pds transcript accumulation in developing endosperm showed Pds was constitutively expressed. Therefore, endosperm carotenogenesis is not regulated by increasing the level of Pds transcripts.

Abstract: We are becoming increasingly aware of the role that translational control plays in regulating gene expression in plants. There are now many examples in which specific mechanisms have evolved at the translational level that directly impact the amount of protein produced from an mRNA. All regions of an mRNA, i.e., the 5′ leader, the coding region, and the 3′-untranslated region, have the potential to influence translation. The 5′-terminal cap structure and the poly(A) tail at the 3′ terminus serve as additional elements controlling translation. Many viral mRNAs have evolved alternatives to the cap and poly(A) tail that are functionally equivalent. Nevertheless, for both cellular and viral mRNAs, a co-dependent interaction between the terminal controlling elements appears to be the universal basis for efficient translation.

Abstract: Arabinogalactan proteins (AGPs) are found in higher plants and in liverworts [5, 10]. The AGPs are proteoglycans occurring on cell membranes, in the extracellular matrix and in gum exudates. One of the best known AGPs is gum arabic which is excreted by Acacia senegal upon wounding. Already some centuries ago, gum arabic was produced in eastern Africa and traded by Arabs. Nowadays gums, containing similar AGPs as gum arabic or AGP-related compounds, are still being used in the food industry as additives for their aggregating and gelling capacity. AGPs do not have a structural function in that they are not covalently linked in the cell wall, and are often secreted in large amounts, for instance in gums. In the latter case they might act as a physical barrier by forming a gel upon wounding, thus preventing attacks by potential pathogens. Since AGPs occur in many different tissues and are not exclusively produced upon wounding, it seems likely that they do have other functions. In this review the role of AGPs in plant differentiation is discussed. Electrophoretic analysis, monoclonal antibodies and addition to cell cultures resulted in circumstantial and direct evidence that AGPs or AGP structures are involved in the development of cells.

Abstract: Glutelin is the most abundant storage protein in rice, which is expressed specifically in the endosperm of maturing seed Glutelin is encoded by about 10 genes per haploid genome, which are clearly divided into two subfamilies (GluA and GluB) Most of them are coordinately expressed during seed maturation in spite of the remarkable divergence in the 5'-flanking regions between members of two subfamilies In order to identify the common regulatory mechanisms responsible for the endosperm-specific expression, various cis-regulatory elements in the 5'-flanking region of the glutelin GluB-1 gene were characterized by studying the expression of chimeric genes that consisted of the sequentially deleted or mutagenized promoter and a beta-glucuronidase (GUS) reporter gene in transgenic tobacco seeds The essential cis-regulatory elements governing the spatially and temporally specific expression of the glutelin gene expression were located within the first 245 bp of the promoter region of the GluB-1 gene from the site of initiation of transcription The AACA motif between positions -73 and -61 common to all the six genes for glutelin sequenced to date and is repeated between positions -212 and -200 is implicated in the seed-specific expression The GCN4 motif between positions -165 and -158 and between positions -96 and -92 that is conserved at homologous sites in all the members of glutelin gene family is also involved in the seed-specific regulation However, both are required for the high level of seed-specific expression, because deletion of the region containing one set of both elements or substitution mutation of the AACA or GCN4 motif substantially reduced the activity As a whole, our results suggest the combinatorial interaction of the elements in regulation of the glutelin gene expression

Abstract: Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.

Abstract: Protein kinases are important in eukaryotic signal transduction pathways. In this study we designed degenerate oligonucleotides corresponding to two conserved regions of protein kinases and using the polymerase chain reaction (PCR) have amplified a 141 bp fragment of DNA from mungbeans (Vigna radiata Rwilcz cv. Berken). Sequence analysis of the PCR products indicates that they encode several putative protein kinases with respect to their identity with other known plant protein kinases. Using one of the six fragments (CPK3-8), we isolated a 2022 bp cDNA (VrCDPK-1) from a Vigna radiata lambda gt11 library. VrCDPK-1 has a 96 bp 5'-untranslated region and a 465 bp 3'-untranslated region and an open reading frame of 1461 bp. VrCDPK-1 contains all of the conserved regions commonly found in calcium dependent protein kinases (CDPK). VrCDPK-1 shares 24 to 89% sequence identity with previously reported sequences for plant CDPKs at the protein level. Southern analysis revealed the presence of several copies of the CDPK gene. VrCDPK-1 expression was stimulated when mungbean cuttings were treated with CaCl2, while treatment with MgCl2 had no effect. We are reporting for the first time a CDPK gene in mungbean which is inducible by mechanical strain. Cuttings treated with indole-3-acetic acid (IAA) or subjected to salt stress showed an increase in VrCDPK-1 expression. There was a dramatic stimulation in VrCDPK-1 expression 6 h after cuttings were treated with cycloheximide.

Abstract: Analysis of Agrobacterium-transferred DNA (T-DNA) revealed strong correlations between transgene structures and floral pigmentation patterns from chalcone synthase (chs) co-suppression among 47 Petunia transformants. Presented here are the full details of T-DNA structural organization in that population. Sixteen transformants (34%) carried one T-DNA copy while 31 (66%) carried 106 complete and partial T-DNA elements in 54 linkage groups. Thirty linkage groups contained multiple T-DNA copies; 15 of these contained only contiguously repeated copies, 8 contained only dispersed copies and 7 contained both. Right-border inverted repeats were three times more frequent than left-border inverted or direct repeats. Large fragments of binary-vector sequences were linked to the T-DNA in seven plants.

Abstract: A putative α-glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A3 (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal α-glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI α-amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this α-glucosidase gene and two additional sequences that may be related genes or pseudogenes.

Abstract: Expression of the maize (Zea mays L.) starch branching enzyme (SBE) genes Sbe1 and Sbe2 were characterized during kernel development and in vegetative tissues. The onset of Sbe1 and Sbe2 expression during endosperm development was similar to that of other genes involved in starch biosynthesis (Wx, Sh2 and Bt2). However, the expression of Sbe2 peaked earlier than that of Sbe1 in developing endosperm and embryos resulting in a shift in the ratio of Sbe1 to Sbe2 relative message levels during kernel and embryo development. Transcripts hybridizing to the Sbe2 probe were not detectable in leaves or roots which nonetheless have SBEII enzymatic activity, suggesting that there may be another divergent SBEII-like gene(s) in maize. A similar expression pattern is shared between the maize genes and related genes in pea, which together with their evolutionary conservation, suggests that the SBE isoforms may play unique roles in starch biosynthesis during plant development.

Abstract: Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whether the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. This increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts sinergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.