Showing papers in "Plant Molecular Biology in 1996"
TL;DR: This review summarizes the current understanding of how molecular chaperones function in plants, with a major focus on those systems where the most detailed mechanistic data are available, or where features of the chaperone/foldase system or substrate proteins are unique to plants.
Abstract: Protein folding in vivo is mediated by an array of proteins that act either as ‘foldases’ or ‘molecular chaperones’. Foldases include protein disulfide isomerase and peptidyl prolyl isomerase, which catalyze the rearrangement of disulfide bonds or isomerization of peptide bonds around Pro residues, respectively. Molecular chaperones are a diverse group of proteins, but they share the property that they bind substrate proteins that are in unstable, non-native structural states. The best understood chaperone systems are HSP70/DnaK and HSP60/GroE, but considerable data support a chaperone role for other proteins, including HSP100, HSP90, small HSPs and calnexin. Recent research indicates that many, if not all, cellular proteins interact with chaperones and/or foldases during their lifetime in the cell. Different chaperone and foldase systems are required for synthesis, targeting, maturation and degradation of proteins in all cellular compartments. Thus, these diverse proteins affect an exceptionally broad array of cellular processes required for both normal cell function and survival of stress conditions. This review summarizes our current understanding of how these proteins function in plants, with a major focus on those systems where the most detailed mechanistic data are available, or where features of the chaperone/foldase system or substrate proteins are unique to plants.
596 citations
TL;DR: The degree ofcosuppression observed in progeny of transgenotes carrying multiple, dispersed copies as compared to that observed with a single copy of the transgene suggests that sense cosuppression ofChs is subject to a transgenes dosage effect.
Abstract: Flower pigmentation patterns were scored in 185 senseChalcone synthase (Chs) transgenotes and 85 antisenseChs transgenotes; upon first flowering, 139 (75%) of sense transgenotes were found to be phenotypically altered, as were 70 (82%) of the antisense transgenotes. The observed patterns document the range of phenotypic variations that occur, as well as confirm and extend the finding that senseChs constructs produce several types of morphologybased based flower pigmentation patterns that antisenseChs constructs do not. Long-term monitoring for epigenetic variations in one population of 44 senseChs transgenotes showed that 43 (98%) were capable of producing a cosuppression phenotype. The primary determinant of sense-specific patterns of cosuppression ofChs was found to be the repetitiveness and organization pattern of the transgene, not ‘position effects’ by, or ‘readthrough’ from, flanking plant DNA sequences. The degree of cosuppression observed in progeny of transgenotes carrying multiple, dispersed copies as compared to that observed with a single copy of the transgene suggests that sense cosuppression ofChs is subject to a transgene dosage effect.
433 citations
TL;DR: From insights into this and other proteolytic pathways, the use of phosphorylation/dephosphorylation and/or the addition of amino acid tags to selectively mark proteins for degradation have become recurring themes.
Abstract: Proteolysis is essential for many aspects of plant physiology and development. It is responsible for cellular housekeeping and the stress response by removing abnormal/misfolded proteins, for supplying amino acids needed to make new proteins, for assisting in the maturation of zymogens and peptide hormones by limited cleavages, for controlling metabolism, homeosis, and development by reducing the abundance of key enzymes and regulatory proteins, and for the programmed cell death of specific plant organs or cells. It also has potential biotechnological ramifications in attempts to improve crop plants by modifying protein levels. Accumulating evidence indicates that protein degradation in plants is a complex process involving a multitude of proteolytic pathways with each cellular compartment likely to have one or more. Many of these have homologous pathways in bacteria and animals. Examples include the chloroplast ClpAP protease, vacuolar cathepsins, the KEX2-like proteases of the secretory system, and the ubiquitin/26S proteasome system in the nucleus and cytoplasm. The ubiquitin-dependent pathway requires that proteins targeted for degradation become conjugated with chains of multiple ubiquitins; these chains then serve as recognition signals for selective degradation by the 26S proteasome, a 1.5 MDa multisubunit protease complex. The ubiquitin pathway is particularly important for developmental regulation by selectively removing various cell-cycle effectors, transcription factors, and cell receptors such as phytochrome A. From insights into this and other proteolytic pathways, the use of phosphorylation/dephosphorylation and/or the addition of amino acid tags to selectively mark proteins for degradation have become recurring themes.
382 citations
TL;DR: White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.
Abstract: The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.
355 citations
TL;DR: In this article, the cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP 2) have been characterized and it was found that AMPs are processed from a precursor preprotein and preproprotein, respectively.
Abstract: The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolle et al., Plant Mol Biol 28:713-721 (1995) and 22:1187-1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2 wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 (1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing either wild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. The in vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with either Botrytis cinerea or Alternaria longipes.
330 citations
TL;DR: To determine which of these models is correct it will be necessary to find out whether transgene methylation, which is frequently associated with the potential of transgenes to confer post-transcriptional gene silencing, is a cause or a consequence of the process.
Abstract: Post-transcriptional gene silencing in transgenic plants is the manifestation of a mechanism that suppresses RNA accumulation in a sequence-specific manner. The target RNA species may be the products of transgenes, endogenous plant genes or viral RNAs. For an RNA to be a target it is necessary only that it has sequence homology to the sense RNA product of the transgene. There are three current hypotheses to account for the mechanism of post transcriptional gene silencing. These models all require production of an antisense RNA of the RNA targets to account for the specificity of the mechanism. There could be either direct transcription of the antisense RNA from the transgene, antisense RNA produced in response to over expression of the transgene or antisense RNA produced in response to the production of an aberrant sense RNA product of the transgene. To determine which of these models is correct it will be necessary to find out whether transgene methylation, which is frequently associated with the potential of transgenes to confer post-transcriptional gene silencing, is a cause or a consequence of the process.
324 citations
TL;DR: The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals and showed that both the secretory signal and the ER retention signal were recognized in the plant cells.
Abstract: The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.
299 citations
TL;DR: Mutation of the core pentamer of two putative low temperature responsive elements in the 5′-proximal region of the winter Brassica napus cold-induced gene BN115 revealed the loss of low-temperature regulation by the promoter, indicating that the CCGAC sequence is critical to the low-Temperature response in the BN 115 gene.
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.
288 citations
TL;DR: The properties of higher-plant introns and trans-acting spliceosomal components are document and the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing are discussed.
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.
281 citations
TL;DR: The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thalina, and three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region.
Abstract: Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da. From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern analysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca. 0.8 nkat per mg of protein. The cDNA encoding A. thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thalina.
267 citations
TL;DR: The cDNA which encodes lycopene cyclase, CrtL, was cloned from tomato and tobacco and functionally expressed in Escherichia coli, supporting the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis.
Abstract: The cDNA which encodes lycopene cyclase, CrtL, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN) and functionally expressed in Escherichia coli. This enzyme converts lycopene to β-carotene by catalyzing the formation of two β-rings at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8′) position. Inhibition in E. coli indicated that lycopene cyclase is the target site for the inhibitor MPTA, 2-(4-methylphenoxy)tri-ethylamine hydrochloride. The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but different from that of from the non-photosynthetic bacteria Erwinia. mRNA of CrtL and Pds, which encodes phytoene desaturase, was measured in leaves, flowers and ripening fruits of tomato. In contrast to genes which encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the ‘breaker’ stage of fruit ripening, the level of CrtL mRNA decreases at this stage. Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis.
TL;DR: The most important results obtained in this study are presented, and are extended to the case of plant D-type cyclins, in which three groups are identified.
Abstract: The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins [1]. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3.
TL;DR: The current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are considered.
Abstract: Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features. Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels. This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression. Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization. Several chloroplast genes are interrupted by introns and therefore require processing for gene function. In tobacco chrloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes. A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chrloroplasts. Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years. Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.
TL;DR: It is revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence, and 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 the mRNA level is maintained at a comparable level in naturally senescing leaves.
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.
TL;DR: The various methods used to optimize expression of a foreign gene in plants by concentrating on post-transcriptional events are discussed.
Abstract: Introducing a foreign gene into a new plant host does not always result in a high level of expression of the incoming gene. Numerous promoters have been used to express foreign genes in different plant tissues, but there are sometime various features of the new gene which are deleterious to expression in the new host. There are a number of post-transcriptional steps in the expression of a gene and sometimes sequences present in a particular coding region can resemble the signals which initiate these processing steps. When aberrantly carried out, these steps diminish the level of expression. By removing such fortuitous signals, one can dramatically increase expression of a transgene in plants. Ensuring proper protein folding and/or targeting the protein product to a particular cellular compartment can also be used to increase the level of protein obtained. The various methods used to optimize expression of a foreign gene in plants by concentrating on post–transcriptional events are discussed.
TL;DR: This review focuses on the current knowledge of protein synthesis in higher plants and its role in eukaryotic and prokaryotic cells.
Abstract: Protein synthesis in both eukaryotic and prokaryotic cells is a complex process requiring a large number of macromolecules: initiation factors, elongation factors, termination factors, ribosomes, mRNA, amino-acylsynthetases and tRNAs. This review focuses on our current knowledge of protein synthesis in higher plants.
TL;DR: 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, suggesting a near constitutive pattern of expression.
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.
TL;DR: Various nuclear and chloroplast mutants of Chlamydomonas deficient in photosynthetic activity have been isolated and characterized, and a set of RNA-binding proteins that interact with the 5′-untranslated regions of plastid mRNAs are identified.
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.
TL;DR: It is hypothesized that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleur one activities during germination and during protoplast isolation.
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.
TL;DR: In mitochondria, recent experiments indicate that RNA editing may be a deamination process, and a plastid transformation technique showed to be a powerful tool for the study of RNA editing is compared.
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.
TL;DR: Findings suggest that some PR genes, as well as being induced by pathogen infection, may have alternative functions during plant development, for example in the process of leaf senescence.
Abstract: Genes that are expressed during leaf senescence in Brassica napus were identified by the isolation of representative cDNA clones. DNA sequence and deduced protein sequence from two senescence-related cDNAs, LSC94 and LSC222, representing genes that are expressed early in leaf senescence before any yellowing of the leaves is visible, showed similarities to genes for pathogenesis-related (PR) proteins: a PR-1a-like protein and a class IV chitinase, respectively. The LSC94 and LSC222 genes showed differential regulation with respect to each other; an increase in expression was detected at different times during development of healthy leaves. Expression of both genes was induced by salicylic acid treatment. These findings suggest that some PR genes, as well as being induced by pathogen infection, may have alternative functions during plant development, for example in the process of leaf senescence.
TL;DR: The promoter region (−309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5′ as well as internal deletions fused to the reporter gene GUS and a cis element similar to the (CA)n element, found in other storage protein promoters, was identified.
Abstract: The promoter region (−309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5′ as well as internal deletions fused to the reporter gene GUS (β-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between −309 to −152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position −152 to position −144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region −133 to −120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)n element, found in other storage protein promoters, was identified. This suggest that the (CA)n element is important for conferring seed specificity by serving both as an activator and a repressor element.
TL;DR: Analysis of senescence in transgenic plants deficient in ethylene biosynthesis, in which leaf senescences is delayed, indicated that enhanced accumulation of SENU2 and SENU3 mRNA was similarly delayed but not prevented.
Abstract: Several cDNAs for mRNAs that change in abundance during tomato leaf senescence were isolated. In this paper we report molecular cloning and expression analysis of two cysteine proteases. SENU2 is identical to the cDNA C14 which encodes a cysteine protease previously shown to be expressed in response to extremes of temperature in tomato fruit [43]. SENU3 cDNA clone was 1.2 kb in length and hybridized to a transcript of 1.4 kb which suggested that the clone was not full-length. The missing 5′ end was isolated using rapid amplification of cDNA ends (RACE). Southern blot analysis of tomato genomic DNA indicates that SENU3 is encoded by a single or low copy gene. SENU3 was also shown to have significant homology with known cysteine proteases. These two senescence-associated cysteine proteases are also expressed during other developmental processes, including seed germination, consistent with a role in protein turnover. SENU2 and SENU3 mRNAs were detectable in young fully expanded leaves and increased in abundance with leaf age, reaching a maximum during the later stages of visible leaf senescence. Such a pattern of expression suggests that the onset of leaf senescence is a gradual event. Analysis of senescence in transgenic plants deficient in ethylene biosynthesis, in which leaf senescence is delayed, indicated that enhanced accumulation of SENU2 and SENU3 mRNA was similarly delayed but not prevented.
TL;DR: The recent first isolation of a blue-light photoreceptor, CRY1 (for cryptochrome), from Arabidopsis thaliana by molecular genetic techniques is focused on, primarily as they relate to the newly identified CRY 1Photoreceptor.
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.
TL;DR: The data show that the barley seed peroxiredoxin is encoded by a singlePer1 gene, the first antioxidant belonging to the 1-Cys subgroup shown to be functionally active, and the first perxiredoxin of any kind to be functional described in plants.
Abstract: Antioxidants can remove damaging reactive oxygen species produced as by-products of desiccation and respiration during late embryogenesis, imbibition of dormant seeds and germination. We have expressed a protein, PER1, encoded by the Balem (barley aleurone and embryo) transcript previously called B15C, and show it to reduce oxidative damage in vitro. PER1 shares high similarity to a novel group of thiol-requiring antioxidants, named peroxiredoxins, and represents a subgroup with only one conserved cysteine residue (1-Cys). PER1 is the first antioxidant belonging to the 1-Cys subgroup shown to be functionally active, and the first peroxiredoxin of any kind to be functionally described in plants. The steady state level of the transcript, Per1, homologous to a dormancy-related transcript (pBS128) from bromegrass (Bromus secalinus), increases considerably in imbibed embryos from dormant barley (Hordeum vulgare L.) grains. Our investigations also indicate that Per1 transcript levels are dormancy-related in the aleurone layer of whole grains. In contrast to most seed-expressed antioxidants Per1 disappears in germinating embryos, and in the mature aleurone the transcript is down-regulated by the germinating embryo or by gibberellic acid (GA). Our data show that the barley seed peroxiredoxin is encoded by a single Per1 gene. Possible roles of the PER1 peroxiredoxin in barley grains during desiccation, dormancy and imbibition are discussed.
TL;DR: It is generally concluded that the oil body may facilitate the efficient breakdown of the storage lipid because of its high surface area to volume ratio and thus allow more efficient catabolism.
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].
TL;DR: A model for a plant 3′-end processing complex and hints that some processing factors are conserved in different organisms are proposed, opening promising avenues toward initial characterisation of the trans-acting factors involved in 3″-end formation of mRNAs in higher plants.
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.
TL;DR: 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 duringxylogenesis.
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.
TL;DR: 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: 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.
TL;DR: In this article, two lines of sunflower (Helianthus annuus L.) were selected in the field as drought-tolerant (R1) or drought-sensitive (S1) genotype.
Abstract: We have studied two lines of sunflower (Helianthus annuus L.) selected in the field as drought-tolerant (R1 genotype) or drought-sensitive (S1 genotype). When subjected to drought conditions, the R1 line was able to maintain high leaf water potential longer and wilted later than the S1 line. Therefore, this indicates that R1 tolerance includes a leafadaptive response. By subtractive hybridization, we have isolated six different cDNAs (designated sdi for sunflower drought-induced) corresponding to transcripts accumulated in R1 and S1 leaves during adaptive response. Analysis of transcript accumulation in response to drought in both genotypes suggests a preferential expression of three sdi genes in the tolerant line. Abscisic acid-mediated induction, analysed in R1 leaves, was observed for only four sdi genes. Sequence analysis of six sdi clones revealed that five clones were related to known proteins including non specific lipid transfer proteins (nsLTP), early light-induced proteins (ELIP), 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) or dehydrins, predicted to be involved in a wide range of physiological processes.