Studio funzionale di due geni MADS-box (FaSHP e TM8) coinvolti nello sviluppo di strutture carnose con funzione "frutto"

The fruit is an ovary derived structure, typical of Angiosperms, that is fundamental for the dispersal of seeds in the environment. Fleshy fruits are particularly efficient because they attract frugivorous animals that disperse the seeds in the environment by means of endozoochory. However the production of fleshy structures for seed dispersal is not limited to flowering plants and can be also found in the Gymnosperms. In fact, some Gymnosperm species surround their seeds with fleshy structures which can be considered fruits from a functional point of view, even if they are not ovary derived. The study of the molecular mechanisms regulating fleshy structure development is therefore interesting from an evolutionary point of view. It was recently discovered that some MADS-box genes, already known to be involved in the development of Angiosperm fruits, are also expressed in Gymnosperm fruits (Lovisetto et al., 2012). This suggests a possible conservation of the molecular mechanisms involved in the development of seed dispersing structures. Since the study of Gymnosperm genes is difficult because these plants are recalcitrant to both transformation and regeneration, we decided to study two Angiosperm genes for this thesis work. In particular the strawberry FaSHP and the tomato TM8 genes, whose homologous ones are also expressed in the Gymnosperm fruits, were studied in strawberry and tomato, respectively. FaSHP is a strawberry AGAMOUS-like MADS-box gene, belonging to the PLENA clade, which was isolated following the screening of a red fruit cDNA library and which is particularly expressed during the ripening phase. Considering the already demonstrated important role of other genes of this group during the development of climacteric fleshy fruits, we decided to investigate FaSHP function by means of transient gene silencing and over-expression experiments in strawberry fruits, which are non-climacteric. FaSHP down- and over-expressing fruits showed respectively a delay and an acceleration of the ripening phase, which were confirmed at the molecular level by means of the altered expression of some ripening related genes. These data led to hypothesize a possible role for FaSHP as general regulator of many aspects of the "ripening syndrome" in strawberry. Moreover FaSHP gene expression analysis following fruit treatments with auxin and ABA (key hormonal regulators of strawberry ripening), and the identification of hormone response sequences in the gene promoter, suggested that FaSHP could at least partially mediate these two hormone role on fruit ripening. Therefore the results of this part of the work suggest that FaSHP could be a "master gene" in strawberry fruit ripening. Considering that AGAMOUS-like genes are also expressed during the development of Gymnosperm fruits, which like strawberries are false fruits, it can be supposed that the Gymnosperm genes could have a similar function during the development of the seed surrounding fleshy structures of these plants. TM8 was the first discovered gene of a poorly studied MADS-box gene group. As well as Gymnosperm TM8-like genes the tomato TM8 gene has an ubiquitary gene expression profile, even if it reaches a maximum in the flower. In order to test the gene role we produced transgenic plants over-expressing TM8 and transgenic plants over-expressing the gene as a chimeric repressor. Since transgenic plants over-expressing TM8 produce flowers having dialytic stamens, we hypothesized that this gene could have a role in the development of male reproductive organs. This idea was supported by the gene expression analysis of other floral organ MADS-box identity genes. Besides TM8 possible function during flower development, the phenotypic and molecular analysis of the plants over-expressing TM8 as a chimeric repressor led to hypothesize a possible function also during the development of other plant organs, both vegetative (leaf) and reproductive (fruit). These roles in several steps of the plant life cycle support the idea that TM8-like genes are very ancient and that they originated early during MADS-box gene evolution. Moreover, TM8 possible function during fruit development suggests that also in Gymnosperms, where TM8-like genes are expressed in seed dispersing structures, they could participate in the development of "fruits

Studio funzionale di due geni MADS-box (FaSHP e TM8) coinvolti nello sviluppo di strutture carnose con funzione "frutto"

The fruit is an ovary derived structure, typical of Angiosperms, that is fundamental for the dispersal of seeds in the environment. Fleshy fruits are particularly efficient because they attract frugivorous animals that disperse the seeds in the environment by means of endozoochory. However the production of fleshy structures for seed dispersal is not limited to flowering plants and can be also found in the Gymnosperms. In fact, some Gymnosperm species surround their seeds with fleshy structures which can be considered fruits from a functional point of view, even if they are not ovary derived. The study of the molecular mechanisms regulating fleshy structure development is therefore interesting from an evolutionary point of view. It was recently discovered that some MADS-box genes, already known to be involved in the development of Angiosperm fruits, are also expressed in Gymnosperm fruits (Lovisetto et al., 2012). This suggests a possible conservation of the molecular mechanisms involved in the development of seed dispersing structures. Since the study of Gymnosperm genes is difficult because these plants are recalcitrant to both transformation and regeneration, we decided to study two Angiosperm genes for this thesis work. In particular the strawberry FaSHP and the tomato TM8 genes, whose homologous ones are also expressed in the Gymnosperm fruits, were studied in strawberry and tomato, respectively. FaSHP is a strawberry AGAMOUS-like MADS-box gene, belonging to the PLENA clade, which was isolated following the screening of a red fruit cDNA library and which is particularly expressed during the ripening phase. Considering the already demonstrated important role of other genes of this group during the development of climacteric fleshy fruits, we decided to investigate FaSHP function by means of transient gene silencing and over-expression experiments in strawberry fruits, which are non-climacteric. FaSHP down- and over-expressing fruits showed respectively a delay and an acceleration of the ripening phase, which were confirmed at the molecular level by means of the altered expression of some ripening related genes. These data led to hypothesize a possible role for FaSHP as general regulator of many aspects of the "ripening syndrome" in strawberry. Moreover FaSHP gene expression analysis following fruit treatments with auxin and ABA (key hormonal regulators of strawberry ripening), and the identification of hormone response sequences in the gene promoter, suggested that FaSHP could at least partially mediate these two hormone role on fruit ripening. Therefore the results of this part of the work suggest that FaSHP could be a "master gene" in strawberry fruit ripening. Considering that AGAMOUS-like genes are also expressed during the development of Gymnosperm fruits, which like strawberries are false fruits, it can be supposed that the Gymnosperm genes could have a similar function during the development of the seed surrounding fleshy structures of these plants. TM8 was the first discovered gene of a poorly studied MADS-box gene group. As well as Gymnosperm TM8-like genes the tomato TM8 gene has an ubiquitary gene expression profile, even if it reaches a maximum in the flower. In order to test the gene role we produced transgenic plants over-expressing TM8 and transgenic plants over-expressing the gene as a chimeric repressor. Since transgenic plants over-expressing TM8 produce flowers having dialytic stamens, we hypothesized that this gene could have a role in the development of male reproductive organs. This idea was supported by the gene expression analysis of other floral organ MADS-box identity genes. Besides TM8 possible function during flower development, the phenotypic and molecular analysis of the plants over-expressing TM8 as a chimeric repressor led to hypothesize a possible function also during the development of other plant organs, both vegetative (leaf) and reproductive (fruit). These roles in several steps of the plant life cycle support the idea that TM8-like genes are very ancient and that they originated early during MADS-box gene evolution. Moreover, TM8 possible function during fruit development suggests that also in Gymnosperms, where TM8-like genes are expressed in seed dispersing structures, they could participate in the development of "fruits"Il frutto, definito in termini botanici come struttura che si sviluppa da un ovario fiorale in seguito ad un evento di fecondazione, è una struttura peculiare delle Angiosperme. Esso ha contribuito in modo rilevante al successo riproduttivo di queste piante essendo principalmente deputato al processo di dispersione dei semi che si sviluppano al suo interno. Tuttavia, la necessità di disperdere i semi nell'ambiente non è unica delle Angiosperme, anche le Gimnosperme producendo semi devono affrontare questa problematica. Sebbene esse non presentino fiori, e pertanto non siano in grado di produrre veri frutti, vi sono molte specie che circondano i propri semi con delle strutture carnose che hanno lo scopo di facilitarne la dispersione, e sono quindi simili ai veri frutti da un punto di vista funzionale. Alla luce di queste considerazioni risulta chiaro che studiare i meccanismi che regolano lo sviluppo e la maturazione dei frutti è importante anche sotto il profilo evolutivo. Recentemente è stato scoperto che gli omologhi di alcuni geni MADS-box che controllano lo sviluppo e maturazione dei veri frutti, sono espressi anche nelle strutture carnose associate ai semi di alcune Gimnosperme (Lovisetto et al., 2012), suggerendo una possibile conservazione di parte dei meccanismi molecolari di base che regolano la formazione delle strutture coinvolte nella dispersione dei semi. Poiché la caratterizzazione funzionale di tali geni risulta difficile nelle Gimnosperme, per il presente lavoro si è deciso di focalizzare l'attenzione su due geni di Angiosperme, omologhi dei geni identificati nelle Gimnosperme. In particolare è stata condotta la caratterizzazione funzionale del gene FaSHP di fragola e del gene TM8 di pomodoro. FaSHP è un gene MADS-box, appartenente al clade PLENA della sottofamiglia AGAMOUS, che è stato isolato mediante lo screening di una libreria di cDNA di frutto rosso e che è risultato essere espresso nel frutto con un profilo di espressione maturazione-specifico. Per alcuni geni appartenenti a questa classe è già stato definito un ruolo di regolazione durante la maturazione dei frutti climaterici. Quindi, si è deciso di caratterizzare funzionalmente il gene FaSHP conducendo degli esperimenti di silenziamento e sovra-espressione transiente in frutti di fragola che sono invece non-climaterici. Il rallentamento e l’accelerazione della maturazione riscontrati nei frutti rispettivamente sotto- e sovra-esprimenti il gene, insieme alla modificata espressione di vari geni marcatori del processo di maturazione, suggeriscono che FaSHP possa essere uno dei protagonisti del network molecolare che regola la maturazione del frutto di fragola. Inoltre, l'analisi di espressione del gene in seguito ai trattamenti ormonali con auxina ed ABA (regolatori chiave per la maturazione di fragola) e l'identificazione nel promotore del gene di segnali di risposta ai due ormoni, hanno indotto ad ipotizzare che FaSHP possa fungere, almeno in parte, da mediatore nella regolazione della maturazione da parte di questi fitormoni. I dati ottenuti in questa prima parte del lavoro indicano dunque che FaSHP possa essere un master gene nella maturazione del frutto di fragola. Considerando che geni AGAMOUS-like sono espressi anche durante lo sviluppo delle strutture carnose delle Gimnosperme, e che queste ultime, come il frutto di fragola, sono dei falsi frutti, si può ipotizzare che anche nelle Gimnosperme i geni di tipo AGAMOUS abbiano un ruolo simile a quello definito per FaSHP. TM8 è invece il primo gene ad essere stato isolato nell’ambito di un gruppo di geni MADS-box ad oggi poco studiati. Andando ad analizzare la sua espressione si è osservato come i trascritti siano presenti in modo pressoché ubiquitario nella pianta, in maniera del tutto simile a quanto già definito per i geni TM8-like delle Gimnosperme. In pomodoro, tuttavia, i trascritti raggiungono un massimo nel fiore. Per far luce sul ruolo di TM8 sono state prodotte piante transgeniche che sovra-esprimono il gene e piante che invece lo sovra-esprimono nella forma di repressore trascrizionale. La presenza di stami dialitici nelle piante sovra-esprimenti TM8 ha indotto a supporre un possibile ruolo del gene nel terzo verticillo fiorale, ipotesi supportata anche dall'analisi di espressione di altri geni MADS-box di identità fiorale. Oltre alla possibile funzione durante lo sviluppo del fiore, l'analisi fenotipica e molecolare delle piante sovra-esprimenti il gene nella forma di repressore trascrizionale ha portato ad ipotizzare un possibile coinvolgimento di TM8 anche nello sviluppo di altri organi della pianta, sia vegetativi (foglia) che riproduttivi (frutto). Questa funzione del gene in varie fasi del ciclo vitale della pianta supporta l'idea che i geni di tipo TM8 siano geni antichi, originatisi precocemente durante l'evoluzione dei geni MADS-box. Inoltre, il coinvolgimento del gene TM8 di pomodoro nello sviluppo del frutto suggerisce che anche nelle Gimnosperme, dove tali geni sono espressi in modo simile nelle strutture carnose deputate alla disseminazione, essi possano intervenire durante lo sviluppo dei "frutti

A SHATTERPROOF-like gene controls ripening in nonclimacteric strawberries, and auxin and abscissic acid antagonistically affect its expression

Strawberries (Fragaria 7ananassa) are false fruits the ripening of which follows the non-climacteric pathway. The role played by a C-type MADS-box gene [SHATTERPROOF-like (FaSHP)] in the ripening of strawberries has been studied by transiently modifying gene expression through either over-expression or RNA-interference-mediated down-regulation. The altered expression of the FaSHP gene caused a change in the time taken by the over-expressing and the down-regulated fruits to attain the pink stage, which was slightly shorter and much longer, respectively, compared to controls. In parallel with the modified ripening times, the metabolome components and the expression of ripening-related genes also appeared different in the transiently modified fruits. Differences in the response time of the analysed genes suggest that FaSHP can control the expression of ripening genes either directly or indirectly through other transcription factor-encoding genes. Because fleshy strawberries are false fruits these results indicate that C-type MADS-box genes like SHATTERPROOF may act as modulators of ripening in fleshy fruit-like structures independently of their anatomical origin. Treatment of strawberries with either auxin or abscissic acid had antagonistic impacts on both the expression of FaSHP and the expression of ripening-related genes and metabolome components

Characterization of TM8, a MADS-box gene expressed in tomato flowers

Background.The identity of flower organs is specified by various MIKC MADS-box transcription factors which act in a combinatorial manner. TM8 is a MADS-box gene that was isolated from the floral meristem of a tomato mutant more than twenty years ago, but is still poorly known from a functional point of view in spite of being present in both Angiosperms and Gymnosperms, with some species harbouring more than one copy of the gene. This study reports a characterization of TM8 that was carried out in transgenic tomato plants with altered expression of the gene. Results. Tomato plants over-expressing either TM8 or a chimeric repressor form of the gene (TM8:SRDX) were prepared. In the TM8 up-regulated plants it was possible to observe aberrant stamens with poorly viable pollen and altered expression of several floral identity genes, among them B-, C- and E-function ones, while no apparent morphological modifications were visible in the other whorls. Oblong ovaries and fruits, that were also parthenocarpic, were obtained in the plants expressing theTM8:SRDX repressor gene. Such ovaries showed modified expression of various carpel-related genes. No apparent modifications could be seen in the other flower whorls. The latter plants had also epinastic leaves and malformed flower abscission zones. By using yeast two hybrid assays it was possible to show that TM8 was able to interact in yeast with MACROCALIX. Conclusions. The impact of the ectopically altered TM8 expression on the reproductive structures suggests that this gene plays some role in the development of the tomato flower. MACROCALYX, a putative A-function MADS-box gene, was expressed in all the four whorls of fully developed flowers, and showed quantitative variations that were opposite to those of TM8 in the aberrant stamens and ovaries. Since the TM8 protein interacted in vitro only with A-function MADS-box proteins like the tomato MACROCALYX, it seems that for the correct differentiation of the tomato reproductive structures possible interactions between TM8 and MACROCALYX proteins might be important