Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes.
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The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented. (+info)
Enhancer-like properties of an RNA element that modulates Tombusvirus RNA accumulation.
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Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3'-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels ( approximately 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus. (+info)
Posttranslational removal of the carboxyl-terminal KDEL of the cysteine protease SH-EP occurs prior to maturation of the enzyme.
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SH-EP is a cysteine protease from germinating mung bean (Vigna mungo) that possesses a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In order to examine the function of the ER retention sequence, we expressed a full-length cDNA of SH-EP and a minus-KDEL control in insect Sf-9 cells using the baculovirus system. Our observations on the synthesis, processing, and trafficking of SH-EP in Sf-9 cells suggest that the KDEL ER-retention sequence is posttranslationally removed either while the protein is still in the ER or immediately after its exit from the ER, resulting in the accumulation of proSH-EP minus its KDEL signal. It is this intermediate form that appears to progress through the endomembrane system and is subsequently processed to form mature active SH-EP. The removal of an ER retention may regulate protein delivery to a functional site and present an alternative role for ER retention sequences in addition to their well established role in maintaining the protein composition of the ER lumen. (+info)
Cucumber cotyledon lipoxygenase during postgerminative growth. Its expression and action on lipid bodies.
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In cucumber (Cucumis sativus), high lipoxygenase-1 (LOX-1) activity has been detected in the soluble fraction prepared from cotyledons of germinating seeds, and the involvement of this enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka [1992] Plant Sci 85: 23-32; I. Fuessner, C. Wasternack, H. Kindl, H. Kuhn [1995] Proc Natl Acad Sci USA 92: 11849-11853). In this study we have investigated the expression of the gene lox-1, corresponding to the LOX-1 enzyme. LOX-1 expression is highly coordinated with that of a typical glyoxysomal enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts accumulated in tissue during in vitro senescence, no accumulation of lox-1 mRNA could be observed, suggesting that lox-1 plays a specialized role in fat mobilization. LOX-1 is also known to be a major lipid body protein. The partial peptide sequences of purified LOX-1 and lipid body LOX-1 entirely coincided with that deduced from the lox-1 cDNA sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are derived from a single gene and that LOX-1 can exist both in the cytosol and on the lipid bodies. We constructed an in vitro oxygenation system to address the mechanism of this dual localization and to investigate the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on the lipids in intact lipid bodies, although degradation of lipid body proteins, either during seedling growth or by treatment with trypsin, allows lipid bodies to become susceptible to LOX-1. We discuss the role of LOX-1 in fat mobilization and its mechanism of action. (+info)
Regulation of nitrite reductase by light and nitrate in the cotyledons of hot pepper (Capsicum annuum L.).
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Light and nitrate are the major factors regulating the nitrite reductase (NiR) amongst various environmental and metabolic cues in plants. Hot pepper was used to investigate this regulatory mechanism of the NiR gene expression and its dependency on light and nitrate. The major results from this study are: (I) the nir partial clone (581 bp) obtained from hot pepper genomic DNA by degenerative polymerase chain reaction exhibited an amino acid sequence that is highly homologous with other plants. (II) Genomic DNA blot analysis and the NiR electrophoretic assay revealed that a small multigene family encodes NiR, which exists at least in two isoforms. (III) The light-mediated increase of NiR activity is correlated with the nitrate concentration, showing saturation kinetics above 50 mM of nitrate. (IV) Exogenous nitrate was required for the appearance of nir transcripts, but not for the enzyme activity. These results suggest that the gene expression of NiR in hot pepper is determined by the presence of nitrate at the transcriptional level. Furthermore, light has a synergistic effect on the action of nitrate on NiR levels. (+info)
Identification of a cis-regulatory element involved in phytochrome down-regulated expression of the pea small GTPase gene pra2.
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The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5'-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5'- and internal deletion analyses revealed that the 93-bp sequence between -734 and -642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression. (+info)
Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens.
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A cDNA clone encoding the Prx7 peroxidase from barley (Hordeum vulgare L.) predicted a 341-amino acid protein with a molecular weight of 36,515. N- and C-terminal putative signal peptides were present, suggesting a vacuolar location of the peroxidase. Immunoblotting and reverse-transcriptase polymerase chain reaction showed that the Prx7 protein and mRNA accumulated abundantly in barley coleoptiles and in leaf epidermis inoculated with powdery mildew fungus (Blumeria graminis). Two isoperoxidases with isoelectric points of 9.3 and 7.3 (P9.3 and P7.3, respectively) were purified to homogeneity from barley coleoptiles. P9.3 and P7.3 had Reinheitszahl values of 3.31 and 2.85 and specific activities (with 2,2'-azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid], pH 5.5, as the substrate) of 11 and 79 units/mg, respectively. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry peptide analysis identified the P9. 3 peroxidase activity as due to Prx7. Tissue and subcellular accumulation of Prx7 was studied using activity-stained isoelectric focusing gels and immunoblotting. The peroxidase activity due to Prx7 accumulated in barley leaves 24 h after inoculation with powdery mildew spores or by wounding of epidermal cells. Prx7 accumulated predominantly in the epidermis, apparently in the vacuole, and appeared to be the only pathogen-induced vacuolar peroxidase expressed in barley tissues. The data presented here suggest that Prx7 is responsible for the biosynthesis of antifungal compounds known as hordatines, which accumulate abundantly in barley coleoptiles. (+info)
Blue light-directed destabilization of the pea Lhcb1*4 transcript depends on sequences within the 5' untranslated region.
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Pea seedlings grown in continuous red light accumulate significant levels of Lhcb1 RNA. When treated with a single pulse of blue light with a total fluence >10(4) micromol m(-2), the rate of Lhcb1 transcription is increased, whereas the level of Lhcb1 RNA is unchanged from that in control seedlings. This RNA destabilization response occurs in developing leaves but not in the apical bud. The data presented here indicate that the same response occurs in the cotyledons of etiolated Arabidopsis seedlings. The blue light-induced destabilization response persists in long hypocotyl hy4 and phytochrome phyA, phyB, and hy1 mutants as well as in far-red light-grown seedlings, indicating that neither CRY1 (encoded by the hy4 locus) nor phytochrome is the sole photoreceptor. Studies with transgenic plants indicate that the destabilization element in the pea Lhcb1*4 transcript resides completely in the 5' untranslated region. (+info)