Characterization of an AGAMOUS gene expressed throughout development of the fleshy fruit-like structure produced by Ginkgo biloba around its seeds.

BACKGROUND: The involvement of MADS-box genes of the AGAMOUS lineage in the formation of both flowers and fruits has been studied in detail in Angiosperms. AGAMOUS genes are expressed also in the reproductive structures of Gymnosperms, yet the demonstration of their role has been problematic because Gymnosperms are woody plants difficult to manipulate for physiological and genetic studies. Recently, it was shown that in the gymnosperm Ginkgo biloba an AGAMOUS gene was expressed throughout development and ripening of the fleshy fruit-like structures produced by this species around its seeds. Such fleshy structures are evolutionarily very important because they favor the dispersal of seeds through endozoochory. In this work a characterization of the Ginkgo gene was carried out by over-expressing it in tomato. RESULTS: In tomato plants ectopically expressing the Ginkgo AGAMOUS gene a macroscopic anomaly was observed only in the flower sepals. While the wild type sepals had a leaf-like appearance, the transgenic ones appeared connately adjoined at their proximal extremity and, concomitant with the development and ripening of the fruit, they became thicker and acquired a yellowish-orange color, thus indicating that they had undergone a homeotic transformation into carpel-like structures. Molecular analyses of several genes associated with either the control of ripening or the ripening syndrome in tomato fruits confirmed that the transgenic sepals behaved like ectopic fruits that could undergo some ripening, although the red color typical of the ripe tomato fruit was never achieved. CONCLUSIONS: The ectopic expression of the Ginkgo AGAMOUS gene in tomato caused the homeotic transformation of the transgenic sepals into carpel-like structures, and this showed that the gymnosperm gene has a genuine C function. In parallel with the ripening of fruits the related transgenic sepals became fleshy fruit-like structures that also underwent some ripening and such a result indicates that this C function gene might be involved, together with other gens, also in the development of the Ginkgo fruit-like structures. It seems thus strengthened the hypothesis that AGAMOUS MADS-box genes were recruited already in Gymnosperms for the development of the fleshy fruit habit which is evolutionarily so important for the dispersal of seeds.

Characterization of a bZIP gene highly expressed during ripening of the peach fruit.

A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.

[Usefulness of databases of general practitioners: the study of chronic cardiovascular diseases in a health district of Turin, Italy]. / Utilità delle informazioni raccolte tramite medici di medicina generale: lo studio delle malattie cardiovascolari croniche in un distretto sanitario torinese.

BACKGROUND: The aim of this study was to evaluate the prevalence of chronic cardiovascular diseases in the territory of a health district in Turin (Italy) to achieve better management of healthcare services according to healthcare needs. METHODS: In order to assess healthcare needs, we surveyed the prevalence of five cardiovascular chronic diseases in the general population, using data from the general practitioners of one health district. RESULTS: Eighty-six general practitioners participated in the study, collecting data regarding 87 885 attendees, for the year 2010. Among men, the following prevalence rates were estimated: 2% prior myocardial infarction, 2.4% other forms of chronic ischemic heart disease, 2% cardiac arrhythmias, 0.4% heart failure, 0.9% late effects of cerebrovascular disease. Prevalence rates were lower among women, except for dysrhythmias and heart failure, and were increasing with age. Prevalence rates were higher than those previously reported by the Italian Cardiovascular Epidemiological Observatory for northwestern regions. In addition, differences in disease distribution were observed among patients when comparing male doctors and female doctors. CONCLUSIONS: Our findings show several critical issues in primary care, suggesting the need for improving the healthcare delivery system within the health district according to local care needs.

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.

PpEG4 is a peach endo-beta-1,4-glucanase gene whose expression in climacteric peaches does not follow a climacteric pattern.

In peach (Prunus persica L. Batsch.) the degradation of the pectic compounds of the cell wall is considered to be the principal component responsible for fruit softening. Many genes encoding enzymes acting on the different polymers of the pectic matrix have been shown to be highly expressed during the late phases of softening, with polygalacturonase being the most important. Nevertheless, it is known that softening starts well before the ethylene climacteric rise which occurs concomitant with the maximal expression of the pectolytic enzymes. The cloning and characterization of PpEG4, an endo-beta-1,4-glucanase (EGase) gene preferentially expressed in preclimacteric fruits, are presented here. PpEG4 belongs to the group of EGases containing, at their carboxy-terminus, a peptide similar to the cellulose binding domain of microbial origin. This EGase is also expressed during abscission of both leaves and fruits. The effect of exogenous ethylene treatments on PpEG4 transcription is null in young fruits and negative in preclimacteric ones, while it is positive in abscission zones. Thus, the expression of PpEG4 seems to be more dependent on the type of separation process rather than being influenced by a direct hormone action. The ability of the PpEG4 regulatory sequences to drive transcription in cells undergoing separation events is also maintained in tomato, where about 3 kb of the gene promoter could drive the expression of gusA in preclimacteric fruits and in the fruit abscission zones.

Different ethylene receptors show an increased expression during the ripening of strawberries: does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits?

Notwithstanding the economic importance of non-climacteric fruits like grape and strawberry, little is known about the mechanisms that regulate their ripening. Up to now no growth regulator has emerged with a primary role similar to that played by ethylene in the ripening of the climacteric fruits. Strawberries can produce ethylene, although in limited amounts. Two cDNAs coding for enzymes of the ethylene biosynthetic pathway (i.e. FaACO1 and FaACO2), and three cDNAs encoding different ethylene receptors have been isolated. Two receptors (i.e. FaEtr1 and FaErs1) belong to the type-I while the third (i.e. FaEtr2) belongs to the type-II group. The expression of both the ACO and the receptor-encoding genes has been studied in fruits at different stages of development and in fruits treated with hormones (i.e. ethylene and the auxin analogue NAA). All the data thus obtained have been correlated to the known data about ethylene production by strawberry fruits. Interestingly, a good correlation has resulted between the expression of the genes described in this work and the data of ethylene production. In particular, similarly to what occurs during climacteric fruit ripening, there is an increased synthesis of receptors concomitant with the increased synthesis of ethylene in strawberries as well. Moreover, the receptors mostly expressed in ripening strawberries are the type-II ones, that is those with a degenerate histidine-kinase domain. Since the latter domain is thought to establish a weaker link to the CTR1 proteins, even the little ethylene produced by ripening strawberries might be sufficient to trigger ripening-related physiological responses.

Isolation and promoter analysis of two genes encoding different endo-beta-1,4-glucanases in the non-climacteric strawberry.

Two endo-beta-1,4-glucanase (EGase; EC 3.2.1.4.) genes, highly expressed during ripening of the non-climacteric strawberries (Fragariaxananassa Duch. cv. Chandler), were isolated. Serial promoter deletions of both genes (i.e. FaEG1 and FaEG3) fused to GUS were transiently assayed in strawberry fruits by using a technique recently developed in this laboratory. Although differences were observed with the short fragments, GUS activity became comparable with the largest fragments of both promoters. The apparently similar strength of the two largest promoter fragments was in contrast with previous results of Northern analyses which demonstrated different transcripts amounts for the two genes. The inclusion of the 3' flanking region of both genes in the transient assays showed that, in the case of FaEG3, the 3' region had a down-regulating effect on the expression of GUS, and this might account for the lower amount of FaEG3 mRNA usually observed in ripe fruits compared to that of FaEG1. Downstream instability elements might be involved in such down-regulation.