Expression of 1-aminocyclopropane-1-carboxylate oxidase during leaf ontogeny in white clover.
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We examined the expression of three distinct 1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf ontogeny in white clover (Trifolium repens). Significant production of ethylene occurs at the apex, in newly initiated leaves, and in senescent leaf tissue. We used a combination of reverse transcriptase-polymerase chain reaction and 3'-rapid amplification of cDNA ends to identify three distinct DNA sequences designated TRACO1, TRACO2, and TRACO3, each with homology to 1-aminocyclopropane-1-carboxylic acid oxidase. Southern analysis confirmed that these sequences represent three distinct genes. Northern analysis revealed that TRACO1 is expressed specifically in the apex and TRACO2 is expressed in the apex and in developing and mature green leaves, with maximum expression in developing leaf tissue. The third gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were raised to each gene product expressed in Escherichia coli, and western analysis showed that the TRACO1 antibody recognizes a protein of approximately 205 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed preferentially in apical tissue. The TRACO2 antibody recognizes a protein of approximately 36.4 kD (as determined by gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that is expressed in the apex and in developing and mature green leaves, with maximum expression in mature green tissue. No protein recognition by the TRACO3 antibody could be detected in senescent tissue or at any other stage of leaf development. (+info)
Differential expression of two novel members of the tomato ethylene-receptor family.
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The phytohormone ethylene regulates many aspects of plant growth, development, and environmental responses. Much of the developmental regulation of ethylene responses in tomato (Lycopersicon esculentum) occurs at the level of hormone sensitivity. In an effort to understand the regulation of ethylene responses, we isolated and characterized tomato genes with sequence similarity to the Arabidopsis ETR1 (ethylene response 1) ethylene receptor. Previously, we isolated three genes that exhibit high similarity to ETR1 and to each other. Here we report the isolation of two additional genes, LeETR4 and LeETR5, that are only 42% and 40% identical to ETR1, respectively. Although the amino acids known to be involved in ethylene binding are conserved, LeETR5 lacks the histidine within the kinase domain that is predicted to be phosphorylated. This suggests that histidine kinase activity is not necessary for an ethylene response, because mutated forms of both LeETR4 and LeETR5 confer dominant ethylene insensitivity in transgenic Arabidopsis plants. Expression analysis indicates that LeETR4 accounts for most of the putative ethylene-receptor mRNA present in reproductive tissues, but, like LeETR5, it is less abundant in vegetative tissues. Taken together, ethylene perception in tomato is potentially quite complex, with at least five structurally divergent, putative receptor family members exhibiting significant variation in expression levels throughout development. (+info)
We sequenced and characterized PMP22 (22-kD peroxisomal membrane protein) from Arabidopsis, which shares 28% to 30% amino acid identity and 55% to 57% similarity to two related mammalian peroxisomal membrane proteins, PMP22 and Mpv17. Subcellular fractionation studies confirmed that the Arabidopsis PMP22 is a genuine peroxisomal membrane protein. Biochemical analyses established that the Arabidopsis PMP22 is an integral membrane protein that is completely embedded in the lipid bilayer. In vitro import assays demonstrated that the protein is inserted into the membrane posttranslationally in the absence of ATP, but that ATP stimulates the assembly into the native state. Arabidopsis PMP22 is expressed in all organs of the mature plant and in tissue-cultured cells. Expression of PMP22 is not associated with a specific peroxisome type, as it is detected in seeds and throughout postgerminative growth as cotyledon peroxisomes undergo conversion from glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased accumulation during the growth of young seedlings, its expression is not stimulated by light. (+info)
Stage- and tissue-specific expression of ethylene receptor homolog genes during fruit development in muskmelon.
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We isolated two muskmelon (Cucumis melo) cDNA homologs of the Arabidopsis ethylene receptor genes ETR1 and ERS1 and designated them Cm-ETR1 (C. melo ETR1; accession no. AF054806) and Cm-ERS1 (C. melo ERS1; accession no. AF037368), respectively. Northern analysis revealed that the level of Cm-ERS1 mRNA in the pericarp increased in parallel with the increase in fruit size and then markedly decreased at the end of enlargement. In fully enlarged fruit the level of Cm-ERS1 mRNA was low in all tissues, whereas that of Cm-ETR1 mRNA was very high in the seeds and placenta. During ripening Cm-ERS1 mRNA increased slightly in the pericarp of fruit before the marked increase of Cm-ETR1 mRNA paralleled climacteric ethylene production. These results indicate that both Cm-ETR1 and Cm-ERS1 play specific roles not only in ripening but also in the early development of melon fruit and that they have distinct roles in particular fruit tissues at particular developmental stages. (+info)
poc1: an Arabidopsis mutant perturbed in phytochrome signaling because of a T DNA insertion in the promoter of PIF3, a gene encoding a phytochrome-interacting bHLH protein.
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The phytochrome family of informational photoreceptors has a central role in regulating light-responsive gene expression, but the mechanism of intracellular signal transduction has remained elusive. In a genetic screen for T DNA-tagged Arabidopsis mutants affected in early signaling intermediates, we identified poc1 (photocurrent 1), which exhibits enhanced responsiveness to red light. This phenotype is absent in a phyB (phytochrome B) null mutant background, indicating that the poc1 mutation enhances phyB signal transduction. The T DNA insertion in poc1 was found to be located in the promoter region of PIF3, a gene encoding a basic helix-loop-helix protein. The mutant phenotype seems to result from insertion-induced overexpression of this gene in red-light-grown seedlings, consistent with PIF3 functioning as a positively acting signaling intermediate. These findings, combined with data from a separate yeast two-hybrid screen that identified PIF3 as a phytochrome-interacting factor necessary for normal signaling, provide evidence that phytochrome signal transduction may include a direct pathway to photoresponsive nuclear genes via physical interaction of the photoreceptor molecules with the potential transcriptional regulator PIF3. (+info)
Transcriptional regulation of plant phosphate transporters.
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Phosphorus is acquired by plant roots primarily via the high-affinity inorganic phosphate (Pi) transporters. The transcripts for Pi transporters are highly inducible upon Pi starvation, which also results in enhanced Pi uptake when Pi is resupplied. Using antibodies specific to one of the tomato Pi transporters (encoded by LePT1), we show that an increase in the LePT1 transcript under Pi starvation leads to a concurrent increase in the transporter protein, suggesting a transcriptional regulation for Pi acquisition. LePT1 protein accumulates rapidly in tomato roots in response to Pi starvation. The level of transporter protein accumulation depends on the Pi concentration in the medium, and it is reversible upon resupply of Pi. LePT1 protein accumulates all along the roots under Pi starvation and is localized primarily in the plasma membranes. These results clearly demonstrate that plants increase their capacity for Pi uptake during Pi starvation by synthesis of additional transporter molecules. (+info)
RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.
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Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors. (+info)
BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize.
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The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript (rbcL) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2-conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize. (+info)