Isolation and characterization of a cDNA coding for cytoplasmic glutamine synthetase of barley.

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Expression of an Endopolygalacturonase Gene During Growth and Abscission of Peach Fruits

Polygalacturonase (PG, EC 3.2.1.15) is one of the cell wall hydrolases involved in the cell separation processes which occur during ripening of some fleshy fruits (FISHER and BENNETT 1991), as well as during abscission of leaves and fruits (HUBERMANN and GOREN 1979; RASC~O et al. 1985; TAYLOR et al. 1990; BONGHI et al. 1992; TAYLOR et al. 1993). In particular, endopolygalacturonase of tomato fruit is the most widely known form of this enzyme, having been characterized at molecular and biochemical level (see literature in ZHENG et al. 1992). Endopolygalacturonase activity has also been found in peach during both abscission and development of fruits, while no such activity could be detected during leaf abscission (BONGHI et al. 1992; ZANCHIN et al. 1993). A few years ago it was observed that polyclonal antibodies raised against a tomato fruit PG (i.e., PGZA) recognized, in soft ripe fruits of peach, a polypeptide with molecular mass similar to that of PG2A. Furthermore, using as a probe a cDNA coding for tomato fruit endopolygalacturonase, the same researchers were able to clone and characterize a 3.5 kb fragment of peach genomic DNA (LEE et al. 1990). On the basis of sequence analysis it was concluded that, besides an unidentified sequence, it contained about the 3' half of a gene which showed, in the coding regions, extensive homology with the tomato PG gene. According to the same researchers, such homology could explain the observed cross-reaction between the antibody to tomato fruit PG and a peach polypeptide, which was therefore suggested to be a peach endopolygalacturonase and the product of the partly characterized gene (LEE et al. 1990). In tomato the gene encoding the fruit endopolygalacturonase seems to be expressed during the fruit ripening, but not during the leaf abscission. In fact, despite a significative rise in PG activity, an antibody to fruit PG did not recognize any leaf abscission protein. Moreover, a cDNA encoding a tomato fruit PG gave no hybridization to mRNA obtained from activated abscission zones of tomato leaves (TAYLOR et al. 1990). In peach it has recently been shown that a cDNA coding for tomato fruit PG hybridized to mRNA obtained from fruit abscission zones but not from leaf ones where, in any case, no PG activity had been detected (BONGHI et al. 1992). In peach, cell separation events which show an involvement of endopolygalacturonase, are not restricted to fruit softening and abscission. Recently, it has been found that PG activity can also be detected throughout the fruit growth ( ZANCHIN et al. in press). On the basis of the above findings we considered it of some interest to see whether the endopolygalacturonase activity, observed in the course of different cell separation events in peach, is due to expression of the partly known PG gene (LEE et al. 1990) or, as already observed in tomato (TAYLOR et al. 1990), only some of that activity can be ascribed to expression of that gene

Characterization of cCe13, a Member of the Pepper Endo‐β‐1, 4‐Glucanase Multigene Family

In pepper plants the enzyme endo-beta-1,4-glucanase (EGase) is encoded by a multigene family. Here is described the characterization of cCel3, a cDNA which codes for the third EGase known so far in this plant. The known members of this family are present as single copy genes, as demonstrated by a Southern analysis of the genomic DNA. Analysis of the expression of cCel3 demonstrates that the highest levels of the cCel3 mRNA are found in abscission zones of leaves and flowers activated by treatments with the plant hormone ethylene. However, the amounts of the cCel3 transcripts (detectable only by RT-PCR) are always much lower than those of cCel2 so that the latter can be regarded as the "abscission" EGase while cCel3 is likely to perform a role ancillary to that of cCel2

A microarray approach to identify genes involved in seed-pericarp cross-talk and development in peach

<p>Abstract</p> <p>Background</p> <p>Field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. In fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. Moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. Later in embryo development, the interactions between seed and fruit development become less strict. As there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the μPEACH 1.0 array platform and quantitative real time RT-PCR (qRT-PCR) was used to study this process.</p> <p>Results</p> <p>A comparative analysis of the transcription profiles conducted in seed and mesocarp (cv Fantasia) throughout different developmental stages (S1, S2, S3 and S4) evidenced that 455 genes are differentially expressed in seed and fruit. Among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. Seed markers were a LTP1 (lipid transfer protein), a PR (pathogenesis-related) protein, a prunin and LEA (Late Embryogenesis Abundant) protein, for S1, S2, S3 and S4, respectively. Mesocarp markers were a RD22-like protein, a serin-carboxypeptidase, a senescence related protein and an Aux/IAA, for S1, S2, S3 and S4, respectively.</p> <p>The microarray data, analyzed by using the HORMONOMETER platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. Results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly (auxin) or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on.</p> <p>Conclusions</p> <p>In this research, genes were identified marking different phases of seed and mesocarp development. The selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits. Regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones.</p> <p>Further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk (i.e. hormone peptides and microRNAs).</p

Next Generation Sequencing of Ancient Fungal Specimens: The Case of the Saccardo Mycological Herbarium

Despite their essential role in the environment, the number of known fungal species is still low compared to the recent estimates of the fungal biodiversity, principally because of their often cryptic or ambiguous morphological traits. Recent studies have reported that the number of fungal DNA sequences deposited in public DNA databases and representing correctly a species appears dramatically low (approx. 120,000) when compared with the estimated total number of species (approx. from 2.2 to 3.8 million). Thus, the linkage of curated DNA sequence data to expertly identified voucher specimens is of fundamental importance to fill the present gap between the different sizes of described and sequenced fungal diversity. To this purpose, the mycological herbarium collections are considered an important source for fungal DNA-barcoding, and collection-based sequencing is a relevant priority for the coming decades. Unfortunately, ancient herbarium samples have both time and conservation related DNA damages, besides exogenous DNA contamination, that make nucleic acid extraction and amplification challenging. Here, we present the results of DNA extraction, ITS2 amplification and Illumina MiSeq sequencing of 36 specimens from the Saccardo Mycological Herbarium that were collected in the late XIX century and assigned to the genus Peziza. High-throughput sequencing was chosen as an alternative to the conventional Sanger- and cloning-based methods to overcome the high fragmentation of the ancient DNA and the massive occurrence of non-target DNA from fungal contaminants. Our approach has permitted to assign ITS2 sequences to 23 out of the 36 specimens studied in this work, thus providing a univocal DNA sequence for those one century old samples. Furthermore, the ITS2 sequence analysis has permitted a taxonomic study of the samples that has resulted in a revaluation of 5 samples at the species level and 18 samples at genus or higher level. Our results highlight the possibility to apply the technique presented in this work also to the old and more precious type specimens in order to relate a DNA sequence to the species distinctive sample, coupling the traditional morphological description of the species with a DNA sequence

A PLENA-like gene of peach is involved in carpel formation and subsequent transformation into a fleshy fruit

MADS-box genes have been shown to play a role in the formation of fruits, both in Arabidopsis and in tomato. In peach, two C-class MADS-box genes have been isolated. Both of them are expressed during flower and mesocarp development. Here a detailed analysis of a gene that belongs to the PLENA subfamily of MADS-box genes is shown. The expression of this PLENA-like gene (PpPLENA) increases during fruit ripening, and its ectopic expression in tomato plants causes the transformation of sepals into carpel-like structures that become fleshy and ripen like real fruits. Interestingly, the transgenic berries constitutively expressing the PpPLENA gene show an accelerated ripening, as judged by the expression of genes that are important for tomato fruit ripening. It is suggested that PpPLENA might interfere with the endogenous activity of TAGL1, thereby activating the fruit ripening pathway earlier compared with wild-type tomato plants

A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium

Fleshy fruits represent a very important economic resource and, therefore, they are an ideal target for biotechnological ameliorations. However, because of their physiological and anatomical characteristics, ripe fleshy fruits represent an extremely difficult material for transient gene expression assays aimed at the study of gene promoters in a short time. To this purpose, a fast and efficient Agrobacterium-mediated transient gene expression system was developed for ripe fleshy fruits. A P-glucuronidase reporter gene interrupted by an intron was used in order to prevent the possible expression of GUS activity by the Agrobacterium cells. The contemporary use of another reporter gene was used to check the transformation efficiency. This method is based on the injection of an Agrobacterium suspension into the fruits, and allows both qualitative and quantitative assays in a wide range of fruits to be carried out