Expression of a polygalacturonase associated with tomato seed germination.

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

Stimulation of homologous recombination in plants by expression of the bacterial resolvase ruvC.

Targeted gene disruption exploits homologous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.

Analysis of the Ac promoter: structure and regulation.

The Ac-encoded transposase, a factor that is essential for the mobility of the Ac element, is expressed under the control of a promoter that lacks a conventional TATA box. The regulation of this promoter is poorly understood. We have analyzed Ac promoter structure and activity, both in vitro and in vivo, using transgenic tobacco plants and cell suspensions. A deletion analysis of the Ac 5' region showed that the minimal promoter is located within 70 bp of the major transcription initiation site (at position 334). The minimal promoter includes the sequence TAAGAAATA at position 294 303, i.e., about 30 nucleotides upstream from the transcription start site. This sequence binds specifically to the TATA-binding protein (TBP), suggesting that it is functional as a TATA box. The regulation of the Ac promoter was studied throughout plant development. Levels of Ac mRNA were low in all tissues studied, with higher expression being observed in dividing cells. In order to test whether Ac promoter is regulated during the cell cycle, a tobacco cell suspension transformed with Ac, was grown synchronously. No differences were found in Ac mRNA levels between cells in S, G2, M, or G1 phases; however, expression was lower in the stationary phase. We conclude that Ac promoter is not cell-cycle regulated but is expressed at a higher level in dividing cells. The possible relationship between promoter features and the regulation of Ac element transposition is discussed.

Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene: A Re-examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 L/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 L/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 L/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 L/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 L/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.

Binding of Nicotiana nuclear proteins to the subterminal regions of the Ac transposable element.

Specific binding of Nicotiana nuclear protein(s) to subterminal regions of the Ac transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions of Ac, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends of Mutator. Moreover, we show that DNA from Ds1 competes for protein binding to Ac termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region of Tgm1, Tpn1, En/Spm, Tam3, and Ds1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.

Cloning and primary structure of the chiA gene from Aeromonas caviae.

The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chitinase A of Serratia marcescens. Expression in Escherichia coli yielded an enzymatically active protein from which a leader sequence was removed, presumably during transport of the enzyme across the cell membrane.

Interference of phenolic compounds with the 1-aminocyclopropane-1-carboxylic Acid assay.

The yields of ethylene from endogenous and exogenous 1-aminocyclo-propane-1-carboxylic acid (ACC) in avocado (Persea Americana Mill.) fruit pedicel extracts were very low when assayed by the method of Lizada and Yang (1979 Anal Biochem 100: 140-145). Addition of phenolic compounds, which are present in avocado tissues, to the assay mixture significantly reduced the conversion efficiency of ACC to ethylene. A negative correlation was found between the amount of the plant material in the assay mixture and the conversion efficiency of ACC to ethylene. Removal of phenolic compounds from pedicel extracts by polyvinylpolypyrrolidone, Amberlite XAD-7, and Dowex-50 column chromatography or lead acetate precipitation greatly increased the yields of thylene from ACC in these extracts. The use of polyvinylpolypyrrolidone column chromatography also enabled us to obtain more accurate estimations of endogenous ACC levels in carnation (Dianthus caryophyllus L.) petal extracts. The conversion efficiency of ACC to ethylene could be improved by increasing the concentrations of mercuric chloride and NaOCl in the assay mixture.

Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening.

Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity.