Biomedical subjects

D Dellapenna

Publications and source records attributed to D Dellapenna.

8 recordsLinked to original sources

PubMed2001

Analysis of carotenoid biosynthetic gene expression during marigold petal development.

Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to >100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties.

Blotting, Northern↗

PubMed2001

Photoprotection in a zeaxanthin- and lutein-deficient double mutant of Arabidopsis.

When light absorption by a plant exceeds its capacity for light utilization, photosynthetic light harvesting is rapidly downregulated by photoprotective thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). To address the involvement of specific xanthophyll pigments in NPQ, we have analyzed mutants affecting xanthophyll metabolism in Arabidopsis thaliana. An npq1 lut2 double mutant was constructed, which lacks both zeaxanthin and lutein due to defects in the violaxanthin de-epoxidase and lycopene in-cyclase genes. The npq1 lut2 strain had normal Photosystem II efficiency and nearly wild-type concentrations of functional Photosystem II reaction centers, but the rapidly reversible component of NPQ was completely inhibited. Despite the defects in xanthophyll composition and NPQ, the npq1 lut2 mutant exhibited a remarkable ability to tolerate high light.

Journal Article↗

PubMed1990

Changes in mRNA and Protein during Ripening in Apple Fruit (Malus domestica Borkh. cv Golden Delicious).

Poly(A)(+) RNA was extracted from cortical tissue of apple fruit (Malus domestica Borkh. cv Golden Delicious), and in vitro translation products were analyzed by two-dimensional gel electrophoresis. As fruit internal ethylene concentration increased from a basal level of 0.0 to 0.1 microliter per liter to 1 to 5 microliters/liter, substantial changes in the pattern of in vitro translation products were observed. More subtle changes were observed as fruit continued to ripen and internal ethylene concentration increased to 80 to 100 microliters/liter. Overall, the levels of at least six mRNAs were found to increase, while one mRNA decreased. Analysis of proteins extracted from ripening fruit indicated that the level of at least three proteins increased with ripening.

Journal Article↗

PubMed1990

Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit.

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.

Journal Article↗

PubMed1989

Transcriptional Analysis of Polygalacturonase and Other Ripening Associated Genes in Rutgers, rin, nor, and Nr Tomato Fruit.

We have studied the transcription of polygalacturonase (PG) and several other riponing-associated genes in wild-type tomato (Lycopersicon esculentum) fruit and three ripening-impaired mutants, rin, nor, and Nr. In wild-type fruit, the PG gene becomes transcriptionally active early in ripening and remains transcriptionally active during the ripening process. Fruit of the three ripening-impaired mutants, which have reduced levels of PG mRNA, have correspondingly reduced PG transcription rates. Other ripening-associated genes showed diverse patterns of expression in the ripening-impaired mutant backgrounds. These results indicate that transcriptional activation of the PG gene is an important control point regulating the expression of PG during ripening in wild-type fruit and that PG expression in rin, nor, and Nr fruit is blocked at the level of transcription. A comparison of PG transcription rates and mRNA levels with those of other ripening-associated genes suggests that posttranscriptional processes may also contribute to the large accumulation of PG mRNA during ripening.

Journal Article↗

PubMed1988

In vitro synthesis and processing of tomato fruit polygalacturonase.

The in vitro processing of tomato fruit polygalacturonase (PG) (poly[1,4-alpha-d-galacturonide]glucanohydrolase, EC 3.2.1.15) was studied. Complete chemical deglycosylation of a mixture of mature, purified PG 2A and PG 2B isozymes (45 and 46 kilodaltons; respectively) with trifluoromethane sulfonic acid yielded a single polypeptide of 42 kilodaltons. Similarly, N-terminal amino acid sequencing of the PG 2A/2B isozyme mixture yielded a single 21 amino acid N-terminal sequence, suggesting that the two isozymes result from differential post-translational processing of a single polypeptide. Translation of PG mRNA in vitro results in the synthesis of a single polypeptide with an apparent molecular weight of 54 kilodaltons. Nucleotide sequence analysis of a full-length PG cDNA clone indicates that the large size difference between the PG in vitro translation product and the mature isozymes is due to the presence of a 71 amino acid (8.2 kilodaltons) domain at the N-terminus of in vitro translated PG, consisting of a hydrophobic signal sequence followed by a highly charged prosequence. To determine the precise cleavage site of the signal sequence, PG mRNA was translated in vitro in the presence of canine pancreas microsomal membranes. This resulted in the production of two glycosylated PG processing intermediates with apparent molecular weights of 58 and 61 kilodaltons. The PG processing intermediates were shown to be sequestered within the lumen of the microsomal membranes by protease protection and centrifugational analysis. Deglycosylation of the PG processing intermediates with endoglycosidase H yielded a single polypeptide with an apparent molecular weight of 54 kilodaltons. The production of two distinct, glycosylated processing intermediates from the single in vitro translated PG polypeptide suggests a mechanism by which the differential glycosylation observed for the mature PG 2A and PG 2B isozymes may occur. Edman degradation of (3)H-labeled 58 and 61 kilodalton PG processing intermediates indicates that the site of signal sequence cleavage is after amino acid 24 (serine). These results suggest that the proteolytic processing of PG occurs in at least two steps, the first being the co-translational removal of the 24 amino acid signal sequence and the second being the presumed post-translational removal of the remaining highly charged 47 amino acid prosequence.

Journal Article↗

PubMed1987

Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits.

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant.

Journal Article↗

PubMed1986

Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening.

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

Journal Article↗