Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth.
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The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment. In 0.3% (v/v) CO2, the mutant and wild-type plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO2-fixing rate and its ability to distinguish between CO2 and O2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O2 concentrations. Consistent with the reduced CO2 fixation rate at saturating CO2, the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed. (+info)
The tomato golden mosaic virus transactivator (TrAP) is a single-stranded DNA and zinc-binding phosphoprotein with an acidic activation domain.
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The AL2 gene found in members of the genus Begomovirus of the Geminiviridae encodes a transcriptional activator protein (TrAP; also known as AL2, AC2, or C2 protein). TrAP activates expression from the viral coat protein (CP) and BR1 movement gene promoters in mesophyll cells and protoplasts and acts to derepress the CP promoter in vascular tissue. The experiments presented here were designed to elucidate some of the biochemical properties of this multifunctional regulatory protein and to define its activation domain. The results indicate that TrAP from tomato golden mosaic virus (TGMV) binds single-stranded DNA in a sequence nonspecific manner and only weakly interacts with double-stranded DNA, confirming earlier results obtained with TrAP from other begomoviruses. In addition, evidence is presented that indicates that TrAP binds zinc and that zinc is necessary for optimal interaction with ssDNA. We also show that TrAP is phosphorylated when expressed in insect cells and that it contains a transcriptional activation domain of the acidic type. The minimal activation domain is quite small; the region comprising only the 15 C-terminal amino acids of the protein is capable of activating transcription in mouse fibroblasts (NIH3T3 cells) when fused to a heterologous DNA-binding domain. (+info)
Suppressors of systemin signaling identify genes in the tomato wound response pathway.
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In tomato plants, systemic induction of defense genes in response to herbivory or mechanical wounding is regulated by an 18-amino-acid peptide signal called systemin. Transgenic plants that overexpress prosystemin, the systemin precursor, from a 35S::prosystemin (35S::prosys) transgene exhibit constitutive expression of wound-inducible defense proteins including proteinase inhibitors and polyphenol oxidase. To study further the role of (pro)systemin in the wound response pathway, we isolated and characterized mutations that suppress 35S::prosys-mediated phenotypes. Ten recessive, extragenic suppressors were identified. Two of these define new alleles of def-1, a previously identified mutation that blocks both wound- and systemin-induced gene expression and renders plants susceptible to herbivory. The remaining mutants defined four loci designated Spr-1, Spr-2, Spr-3, and Spr-4 (for Suppressed in 35S::prosystemin-mediated responses). spr-3 and spr-4 mutants were not significantly affected in their response to either systemin or mechanical wounding. In contrast, spr-1 and spr-2 plants lacked systemic wound responses and were insensitive to systemin. These results confirm the function of (pro)systemin in the transduction of systemic wound signals and further establish that wounding, systemin, and 35S::prosys induce defensive gene expression through a common signaling pathway defined by at least three genes (Def-1, Spr-1, and Spr-2). (+info)
Molecular and biochemical characterization of the involvement of cyclin-dependent kinase A during the early development of tomato fruit.
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Following fruit set, the early development of tomato (Lycopersicon esculentum Mill.) fruit comprises two distinct phases: a cell division phase and a consecutive phase of cell expansion until the onset of ripening. In this study, we analyzed cytological and molecular changes characterizing these early phases of tomato fruit development. First we investigated the spatial and temporal regulation of the mitotic activity during fruit development. The DNA content of isolated nuclei from the different fruit tissues was determined by flow cytometry analysis. The results confirm the data of mitotic activity measurements and show that cell differentiation, leading to expanded cells, is characterized by endoreduplication. Second, we isolated two cDNAs, named Lyces;CDKA1 (accession no. Y17225) and Lyces;CDKA2 (accession no. Y17226), encoding tomato homologs of the cyclin-dependent kinase (CDK) p34(cdc2). Tomato CDKA gene expression was followed at both the transcriptional and translational levels during fruit development. The transcripts for Lyces;CDKA1 and Lyces;CDKA2 and the corresponding CDKA proteins are predominantly accumulated during the phase of cell division between anthesis and 5 d post anthesis (DPA). In whole fruits, the maximum CDK activity was obtained between 5 and 10 DPA. The determination of the kinase activity using protein extracts from the different fruit tissues was in agreement with mitotic activity analysis. It showed the particular disappearance of the activity in the gel tissue as early as 15 DPA. The overall data of CDK activity measurements suggest a strong post-translational regulation of CDK at the temporal and spatial levels during early tomato fruit development. (+info)
The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond.
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A pyridoxal 5'-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet. (+info)
Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato.
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The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species. (+info)
A homolog of old yellow enzyme in tomato. Spectral properties and substrate specificity of the recombinant protein.
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A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) from Arabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein in Escherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest with N-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of the LeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone. Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration with para-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR. (+info)
Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids.
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Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid. (+info)