Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typical systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenic plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced in the recovered tissue. The resistance was virus specific. Recovered plant tissue could not be infected with TEV, but was susceptible to the closely related virus, potato virus Y. The resistance phenotype was functional at the single-cell level because protoplasts from recovered transgenic tissue did not support TEV replication. Surprisingly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studies suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.
Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typlcal systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenlc plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced ln the recovered tissue. The resistance was virus speclflc. Recovered plant tissue could not be lnfected wlth TEV, but was susceptlble to the closely related virus, potato virus Y. The resistance phenotype was functional at the slngle-cell leve1 because protoplasts from recovered transgenic tissue did not support TEV repllcation. Surprlslngly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studles suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular leve1 by a cytoplasmic actlvity that targets speclfic RNA sequences for inactlvation.
SummaryJasmonates, potent lipid mediators of defense gene expression in plants, are rapidly synthesized in response to wounding. These lipid mediators also stimulate their own production via a positive feedback circuit, which depends on both JA synthesis and JA signaling. To date, molecular components regulating the activation of jasmonate biogenesis and its feedback loop have been poorly characterized. We employed a genetic screen capable of detecting the misregulated activity of 13-lipoxygenase, which operates at the entry point of the jasmonate biosynthesis pathway. Leaf extracts from the Arabidopsis fou2 (fatty acid oxygenation upregulated 2) mutant displayed an increased capacity to catalyze the synthesis of lipoxygenase (LOX) metabolites. Quantitative oxylipin analysis identified less than twofold increased jasmonate levels in healthy fou2 leaves compared to wild-type; however, wounded fou2 leaves strongly increased jasmonate biogenesis compared to wounded wild-type. Furthermore, the plants displayed enhanced resistance to the fungus Botrytis cinerea. Higher than wild-type LOX activity and enhanced resistance in the fou2 mutant depend fully on a functional jasmonate response pathway. The fou2 mutant carries a missense mutation in the putative voltage sensor of the Two Pore Channel 1 gene (TPC1), which encodes a Ca 2þ -permeant non-selective cation channel. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. The results indicate that cation fluxes exert strong control over the positive feedback loop whereby JA stimulates its own synthesis.
Pea weevil (Bruchus pisorum L.) oviposition on pods of specific genetic lines of pea (Pisum sativum L.) stimulates cell division at the sites of egg attachment. As a result, tumor-like growths of undifferentiated cells (neoplasms) develop beneath the egg. These neoplasms impede larval entry into the pod. This unique form of induced resistance is conditioned by the Np allele and mediated by a recently discovered class of natural products that we have identified from both cowpea weevil (Callosobruchus maculatus F.) and pea weevil. These compounds, which we refer to as ''bruchins,'' are long-chain ␣,-diols, esterified at one or both oxygens with 3-hydroxypropanoic acid. Bruchins are potent plant regulators, with application of as little as 1 fmol (0.5 pg) causing neoplastic growth on pods of all of the pea lines tested. The bruchins are, to our knowledge, the first natural products discovered with the ability to induce neoplasm formation when applied to intact plants.
The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA 1 , which is formed from GA 20 by 3-hydroxylation. This step, which limits GA 1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3-hydroxylase from lines of pea carrying the Le, le, le-3, and le d alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from le d , a mutant derived from an le line, contains both the le ''mutation'' and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA 9 to GA 4 , and GA 20 to GA 1 , with K m values of 1.5 M and 13 M, respectively. The amino acid substitution in the clone from le increased K m for GA 9 100-fold and reduced conversion of GA 20 to almost nil. Expression products from le and le-3 possessed similar levels of 3-hydroxylase activity, and the expression product from le d was inactive. Our results suggest that the 3-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.
Abscisic acid (ABA) is required for the regulation of seed maturation in maize (Zea mays L.). Mutants blocked in ABA synthesis (such as viviparous-5) do not mature to quiescent, desiccationtolerant seeds, but germinate on the ear midway through kernel development. Because gibberellins (GA) and ABA act antagonistically in many aspects of plant development, we hypothesized that ABA antagonizes a positive GA signal for precocious germination in maize. In these experiments, we show that a GA deficiency early in seed development, induced genetically or via biosynthesis inhibitors, suppresses vivipary in ABA-deficient developing kernels. The resulting seeds have both desiccation tolerance and storage longevity. Temporal analysis of GA accumulation in wild-type kernels revealed the accumulation of bioactive GA 1 and GA 3 prior to the peak in ABA content. We speculate that these GAs stimulate a developmental program leading to vivipary in the absence of normal amounts of ABA, and that a reduction of GA content reestablishes an ABA/GA ratio appropriate for suppression of germination and induction of maturation. In contrast, the induction of a GA deficiency did not suppress vivipary in viviparous-1 mutant kernels, suggesting that VP1 acts downstream of both GA and ABA in programming seed development.
The amount of active gibberellin (GA) in plant tissues is determined in part by its rate of catabolism through oxidation at C-2. In pea (Pisum sativum L.) seeds, GA 2-oxidation is controlled by the SLN (SLENDER) gene, a mutation of which produces seedlings characterized by a slender or hyper-elongated phenotype. We cloned a GA 2-oxidase cDNA from immature pea seeds by screening an expression library for enzyme activity. The clone contained a full-length open reading frame encoding a protein of 327 amino acids. Lysate of bacterial cultures expressing the protein converted the C 19 -GAs, GA 1 , GA 4 , GA 9 , and GA 20 to the corresponding 2-hydroxy products. GA 9 and GA 20 were also converted to GA 51 and GA 29 catabolites, respectively. The gene appeared to be one member of a small family of GA 2-oxidases in pea. Transcript was found predominantly in roots, flowers, young fruits, and testae of seeds. The corresponding transcript from sln pea contained a point mutation and did not produce active enzyme when expressed heterologously. RFLP analysis of a seedling population segregating for SLN and sln alleles showed the homozygous mutant allele co-segregating with the characteristic slender phenotype. We conclude that SLN encodes GA 2-oxidase.
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