In a previous study, a 65 kDa protein, TDI-65, was found to be accumulated in the leaves of drought-stressed tomato (Lycopersicon esculentum cv. Starfire) plants. The protein level returns to control level when the drought-stressed plants are rewatered. Antibodies raised against the purified protein were used to elucidate the subcellular localization of the protein. The protein was found to be mainly localized in the nuclei and chloroplasts of drought-stressed leaf cells. To identify the nature of the protein, a cDNA library was constructed and screened by the purified anti-TDI-65 antibody. A cDNA clone designated tdi-65 was isolated and characterized. The deduced amino acid sequences of tdi-65 protein has extensive homology with known cysteine proteases such as actinidin and papain. Northern blot analysis revealed that tdi-65 mRNA is 10-fold higher in drought-stressed plants as compared to control and rewatered plants. Similar results were observed in the tomato cultivar Ailsa and its near isogenic abscisic acid (ABA)-deficient mutant line, flacca, suggesting that the gene does not require ABA for its expression under drought conditions. Based on the previous immunolocalization findings we suggest that tdi-65 encoded cysteine protease functions in relation to drought-induced senescence and programmed cell death.
The development of different plant organs (root, hypocotyl, and cotyledons) during seed germination is connected with the transformation of proplastids, which are found in embryonic and meristematic tissues, into amyloplasts in root tissues and into chloroplasts in cotyledons. We have analyzed the expression of nuclear and plastid genes coding for the plastid translational apparatus during the first 7 d of Spinacia oleracea development. Results show that the nuclear genes (rpsl, rps22, rp121, and rp140) are expressed from the 1st d of seed imbibition and precede transcription of the chloroplast-encoded genes (photosynthetic and nonphotosynthetic), which starts the 3rd d after the beginning of imbibition. Transcription from the leaf-/cotyledon-specific P1 promoter of the rp121 gene starts on the first imbibition day. lnhibition of chloroplast biogenesis by bleaching in the presence of norflurazon has no influence on the expression from this P1 promoter, suggesting that the onset of transcription of nuclear gene 41/21 is independent of a plastid signal.Plant plastids probably represent the most metamorphic organelles of the living kingdom. Different plastid forms are characterized by tissue-or organ-specific functions such as photosynthesis in chloroplasts of leaves and cotyledons, synthesis and/or storage of starch in amyloplasts of roots and seeds, and carotenoid accumulation in fruit chromoplasts. AI1 plastid types arise during plant development from proplastids, which are small, undifferentiated plastids found in meristematic tissues. They can also originate from the interconversion of plastid types (Schnepf, 1980).In the present paper, we focus on early phases of chloroplast biogenesis during seed imbibition, germination, and seedling growth. Late phases of chloroplast development in leaves have been analyzed at the molecular level (Gruissem, 1989). In particular, the expression of nuclearand chloroplast-encoded photosynthetic genes has been studied during dark to light transition. In contrast, less is known about the molecular level of early phases of chloroplast development. The transcriptionl translation apparatus is synthesized during the early phases of development, as suggested by the presence of numerous ribosomes in etioplasts of dark-grown leaves. Also, it has been shown * Corresponding author; e-mail rmache@bio.grenet.fr; fax 33-76-51-4336, that the conversion of proplastids into chloroplasts is accompanied by high transcription levels of chloroplast genes encoding the transcription/translation apparatus. In contrast, the chloroplast photosynthetic genes are highly expressed only later in development (Bisanz-Seyer et al., 1989;Baumgartner et al., 1993).The nuclear and chloroplast genetic systems participate in a coordinate manner in the synthesis of protein complexes present within plastids. Plastid biogenesis and interconversion are tightly coupled with temporal and spatia1 stages of plant development and are thought to be under the control of both nuclear and plastid genes (Taylor, 1989;Bogorad, 1...
The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PCspecific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.In accordance with the hypothesis that plastids are of endosymbiotic origin most plastid genes are organized into polycistronic transcription units reminiscent of bacterial operons. Plastid rRNA operons show the typical procaryotic gene order of 16 S, 23 S, and 5 S rDNA. These genes are transcribed as large precursor RNAs that are subsequently processed into the various mature rRNA species (1, 2).The promoter regions of plastid rrn operons harbor Escherichia coli-like "-10" and "-35" consensus sequences, like most of the plastid transcription units. These E. coli-like consensus sequences serve as promoter structures (3-5) or as regulatory elements (6, 7). However, the interpretation of results on studies of transcriptional regulation in plastids is complicated by the existence of different types of RNA polymerases. One is nuclear encoded (8 -11) and the other one is encoded on the plastid genome (12-15). The plastid-encoded enzyme can be considered as "E. coli-like" with respect to its subunit composition (16, 17) and promoter usage (6, 18 -20). The composition of the nuclear-encoded RNA polymerase and the promoter structures that are used by this enzyme are not yet clear although several potential transcription start sites for this enzyme have been mapped (5,21,22).In spinach, the rrn operon upstream region contains three different promoter elements (P1, PC, P2), and transcription is thought to be regulated by the transcription factor CDF2 (23). CDF2 acts as a repressor of rRNA transcription by the E. coli-like plastid RNA polymerase and probably as an activator of rRNA transcription by the nuclear-encoded RNA polymerase (6), i.e. rRNA transcription could be regulated exclusively by CDF2. On the other hand, up to now correct initiation at the putative PC start site could not be demonstrated in vitro raising the ...
A cDNA clone encoding a proline-, threonine-, and glycine-rich protein (PTGRP) was isolated from a wild tomato species (Lycopersicon chilense) (L.X. Yu, H. Chamberland, J.G. Lafontain, Z. Tabaeizadeh [1996] Genome 39: 1185-1193). Northern-blot analysis and in situ hybridization studies revealed that PTGRP is downregulated by drought stress. The level of the mRNA in leaves and stems of 8-d drought-stressed plants decreased 5-to 10-fold compared with that in regularly watered plants. The mRNA reaccumulated when drought-stressed plants were rewatered. Antibodies raised against a glutathione S-transferase/PTGRP fusion protein were used to elucidate the subcellular localization of the protein by immunogold labeling. In regularly watered L. chilense plants, PTGRP protein was found to be localized in xylem pit membranes and disintegrated primary walls. Examination of sections from drought-stressed plants revealed a significant decrease in the levels of labeling. In these samples, only a few scattered gold particles were detected in the same areas. In the leaf tissues of plants that had been rewatered for 3 d following an 8-d drought stress, the labeling pattern was similar to that of the regularly watered plants. To our knowledge, PTGRP is the first droughtregulated protein that has been precisely localized in the cell wall.
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