THE MAIZE EMPTY PERICARP MUTANTS, A TOOL TO STUDY THE GENETIC CONTROL OF DEVELOPMENT AND THE INTERACTION BETWEEN NUCLEAR AND MITOCHONDRIAL GENOME.

The empty pericarp4 (emp4) gene encodes a mitochondrion-targeted PentatricoPeptide Repeat (PPR) protein that is necessary for the correct regulation of mitochondrial gene expression in the endosperm. The two main objectives of this thesis are to understand the role exerted by EMP4 during plant development, and to study the action of emp4 at the molecular level. Homozygous mutant emp4 embryos are retarded in their development and unable to germinate; therefore to examine the role of the emp4 gene during seedling development homozygous mutant seedlings were obtained from the cultivation of excised immature embryos on a synthetic medium. When exposed to light, after 30 days of culture, 44% of mutant embryos germinated and only few of them produced a seedling. In contrast, in dark condition mutant embryos have a germination rate of 83% and the number of plants that reached the first and the second leaf stage were almost doubled. A genetic stock carrying an Ac transposon and a double Ds element located on the long arm of chromosome one, where emp4 resides, had been crossed with +/emp4 plants. Chromosome breakages, induced by Ds in somatic tissues, are expected to produce in heterozygous plants clonal sectors hemizygous for the emp4 mutation. Clearly distinguishable yellow sectors were isolated from wild-type green leaves and their hemizygous genotype was confirmed by PCR. Hemyzigous and heterozygous tissues as well as mutant first leaves and primary roots recovered from both light and dark treatment, were analyzed at light microscopy and by TEM analysis. Typical root anatomy with the peculiar root cell layers was observed in both wild-type and in mutant samples, however in mutant epidermis and hypodermis cell layers were less ordered and alterations in the cell shape were evident, particularly in light. Moreover, comparison between mutant and wild-type root morphology, indicated that mutant roots either grown in the dark or exposed to light were less developed then wild-types and the cell layers epidermis and hypodermis were not properly organized. All the mesophyll compartments (epidermis, vessels and parenchyma) were clearly detectable in both mutant and wild-type tissues in both conditions. In the dark, mutant tissues were composed by smaller cells with abnormal shapes, moreover in both genotypes, chloroplasts, that were distinguishable in wild-type leaf grown in light treatment, could not be observed. In the light, mutant leaves were smaller in comparison with wild-type and displayed a pale green color. Abnormalities were founded in sections of homozygous mutant plants, like alteration in cell shape and size, a smaller population of chloroplasts and lack of nuclei. Changes in the subcellular structure were highlighted from the comparison of wild-type and mutant tissues by means of transmission electron microscopy. In emp4 mutant leaves, mitochondria as well as chloroplast populations were significantly reduced and both organelles displayed a less organized structure. In particular, the main alteration were observed in leaf tissues exposed to light while dark-grown tissues seem to be less affected by the effect of emp4 mutation. Many alterations were detected from the comparison of hemyzigous and heterozygous sectors. Such alterations are similar to those observed in homozygous mutant leaf tissues derived from embryo rescue. Thus, we concluded that emp4 gene is essential to leaf tissues in order to develop and maintain a proper cellular organization. These alterations resemble (for some aspects) in part the macro-autophagy model and in part the model for senescence. Light exposure had a deep effect on cell morphology of emp4 cells, as if a magnification in the senescence process was triggered by this signal, particularly visible in the deterioration of the whole cellular components within the mutant cells. Despite the growing number of studies in PPR field the mechanism of action of PPR proteins is still elusive. In addition it is not clear if PPR proteins act alone or with some molecular partners. There are few well documented cases in which PPR proteins are confirmed to be associated in protein complexes where PPR could act as adapters, recruit some additional factors on the RNA target or, in addition, work in a dimeric state like the Arabidopsis HCF152. A complementation test was conducted to address the genetic relationship between emp4 and emp9475 mutants. The test showed controversial results; one gene was inferred from the lack of complementation observed in F1 and two genes based on the observation of a segregation close to the 9 to 7 ratio, expected when the heterozygous emp F1 plants identify two genes. Two hypotheses have been postulated. The first one is that the two mutants are allelic and a third emp gene segregates in the F2/F3 progenies. The second is that the two mutants are ascribable to two linked genes whose product interact. If the second hypothesis were correct, emp9475 would have been a good candidate for the isolation of a partner of emp4. Heterozygous females were crossed with heterozygous or hyperploid B-A translocation males with the aim of establishing the chromosomal arm location of emp9475. The F1 revealed the mutant was obtained from crosses involving the TB-1L-a male parent, thus indicating that emp9475 lies on the long arm of chromosome 1. A more refined position for emp9475 was achieved by analysis of simple sequence repeat (SSR) marker distribution in a segregating population obtained by crossing heterozygous females with B73, Mo17 and LEL (Large Embryo Line) inbred male parents. A polymorphism for the marker bnlg1347 established a distance of about 1 cM (1 recombinant out of 13) between this marker and emp9475 on the long arm of chromosome 1 at 1.10 bin. These data confirmed the linkage between the two genes. Different molecular analyses were performed on emp9475 mutants, however the origin of the mutation was not revealed and its function remains to be elucidated. A second approach was conducted in parallel to elucidate the mechanism of action of EMP4. The full EMP4 protein as well as two fragment of EMP4 comprising the N and C terminal domains respectively were produced in E. coli using pBAD-Thio-TOPO expression vector and LMG194 strain. The amount of the putative EMP4-thioredoxin fusion protein, produced in E. coli, tend to increase during time of induction until the 70 hours. Prolonged inductions (over 70 hours) seem to had no effect on further accumulation of the protein. Low growing temperature (29 \ub0C) and arabinose concentration at 2%, during the induction period, increase the amount of protein produced by the bacteria

Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels

Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) \u2018Oro A\u2019/melting flesh (MF) \u2018Springcrest\u2019 and \u2018Sanguinella\u2019] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit

Evidence of interaction between mutants of different emp genes

In this report we present an analysis of the allelic relationship of nine emp (empty pericarp) mutants showing a drastic reduction in endosperm tissue production. These mutants have different origin, thus representing independent mutational events. Originally they have been isolated in populations carrying an active MuDr or Spm and they all behave as single gene mutants. To establish their allelic relationship we made crosses of each mutant with the others. To perform this test, for each of the pairwise combinations of the nine mutants, pollen of 10-20 plants of a given mutant, whose heterozygous condition was ascertained by selfing, was applied to the silks of plants representing the selfed progeny of +/emp parents of a different emp isolate. The resulting ears were then scored for visual evidence of mutant segregation. If only wild-type seeds are observed in all ears produced by this cross the two mutants are considered not allelic, whereas if some of ears yield mutants in about one-quarter of the seeds this is taken as evidence of allelism. Wild-type seeds are then taken for further test in F2 and F3, the expectation being that ears should be recovered segregating 3 to 1 for the mutant or not segregating. If the F2 obtained by selfing non-mutant plants of the F1 progeny includes ears segregating an excess of mutants (30-40%), this segregation value, approaching a 9 to 7 ratio, is taken as evidence of heterozygosity for two emp mutants in the parental F1 plant, thus defining two genes. The results of this test, are generally concordant in their conclusions. In four cases, however, where enough data have been collected, the results obtained in F1 and in F2/F3 lead to contrasting conclusions, i.e. one gene as inferred from the lack of complementation observed in F1 and two genes based on the observation of a segregation closet to the 9 to 7 ratio expected when the heterozygous emp F1 plants identify two genes. This intriguing result seems to suggest an interaction between different emp mutants. Technically similar events already reported in the literature are referred as second site non-complementation (SSNC). This event can be explained by assuming interaction between two different mutant proteins leading to a toxic product or that the mutant form of one protein sequesters the wild-type form of the other protein into an inactive complex. In addition we observed two cases when the two mutants under test show non-complementation in the F1and 3:1 segregation in the F2. These are the results expected if the two mutants under test are allelic. However in one case (emp 8075 vs emp 9475), where the two mutants had been assigned to different chromosomes by TB-A mapping, the non-complementation of the two mutants in the F1 could be explained by SSNC due to combined haploinsufficiency. These events of SSNC will be further analyzed to test the basis of these unexpected results

Studio dell’espressione di Cinnamil Alcool Deidrogenasi (CADs) in frutti di pesco (Prunus persica [L.] Batsch.) a differente consistenza e colore della polpa

In pesca la consistenza della polpa del frutto (NMF, MF) potrebbe essere influenzata oltre che dall\u2019attivit\ue0 di enzimi di parete (endo\u2010PG) anche dai composti fenolici, prodotti del metabolismo secondario noti per la loro funzione antiossidante e le propriet\ue0 nutrizionali. Fra questi, l\u2019acido ferulico, intermedio della via biosintetica dei fenilpropanoidi, appare implicato nell\u2019irrigidimento delle pareti cellulari sia tramite la formazione di legami labili con la componente polisaccaridica (ponti di\u2010 o poliferulici) sia come precursore dei monolignoli con formazione di un polimero stabile (lignina). Alcuni enzimi di questa complessa via biosintetica potrebbero svolgere un ruolo importante nel determinare le propriet\ue0 qualitative (tessitura) e nutrizionali dei frutti indirizzando gli intermedi (composti fenolici) verso prodotti finali con diversa biodisponibilit\ue0 e funzione. In frutti di pesca di un genotipo NMF \ue8 stata rilevata la presenza di una putativa Cinnamil\u2010Alcool Deidrogenasi (CAD), enzima chiave in una ramificazione della via dei fenilpropanoidi che porta alla sintesi di lignina. Risultati preliminari ottenuti indicano: a) la presenza in pesco di circa 60 putativi geni cad diffusi nel genoma, di cui risultano maggiormente numerosi o espressi quelli sugli scaffold 6 e 8; b) la presenza nei tessuti (mesocarpo, esocarpo) di frutti di diversi genotipi di un\u2019attivit\ue0 CAD totale i cui livelli risultano variare coerentemente con le caratteristiche fenotipiche di consistenza della polpa e con lo sviluppo della colorazione rossa durante il viraggio. Nel presente studio \ue8 stato condotto un confronto fra le sequenze cad6 e cad8 per individuare una regione adatta allo sviluppo di un anticorpo policlonale anti\u2010CAD sc.8. Nel mesocarpo di frutti di pesco sono stati valutati i livelli di isoforme CAD e l\u2019attivit\ue0 CAD e le loro variazioni sono state discusse in relazione al diverso fenotipo di consistenza (NMF e MF) e colore della polpa (\u201cSanguinella\u201d)

Il ruolo delle Cinnamil Alcool Deidrogenasi durante lo sviluppo e la maturazione del frutto di pesco : studio preliminare

I composti fenolici, prodotti del metabolismo secondario delle piante, e presenti negli alimenti di origine vegetale, hanno grande importanza funzionale nella nutrizione umana in quanto dotati di elevate capacit\ue0 antiossidanti ed antiinfiammatorie. In particolare, nell\u2019uomo i livelli sierici degli acidi salicilico e ferulico, sono correlati al consumo di frutta e verdura. In questo contesto, il contributo relativo di alcuni enzimi chiave dei percorsi metabolici della via dei fenilpropanoidi, che portano alla produzione di lignina (cinnamil alcool deidrogenasi: CAD) o di acido ferulico (REF1) a partire dal precursore comune coniferaldeide, potrebbe essere una discriminante nel determinare le caratteristiche finali del frutto come alimento: maggiore durezza della polpa nel caso di una prevalenza di CAD; maggior presenza di acido ferulico, potenziale agente antiossidante ed antiinfiammatorio, nel caso di una prevalenza di REF1. \uc8 stato peraltro ipotizzato che le CAD possano avere anche la funzione fisiologica di ridirezionare il flusso dei metaboliti dalla sintesi di monolignoli a quella di intermedi coinvolti nella formazione di aromi e di pigmenti, tra cui i composti antocianici che conferiscono colore rosso ai tessuti vegetali. \uc8 accertato che le caratteristiche di tessitura della polpa del frutto, importanti nel definirne le qualit\ue0 organolettiche ed il comportamento in post-raccolta, dipendono da meccanismi coinvolgenti la depolimerizzazione dei componenti polisaccaridici di parete, nei quali un ruolo chiave \ue8 giocato dalle endo-PG. Tuttavia in alcuni frutti \ue8 stato riportato come meccanismi di lignificazione possano, nelle ultime fasi di maturazione, influenzare la durezza della polpa. Studi proteomici hanno evidenziato in frutti di pesca Non Melting Flesh con elevata durezza della polpa una maggiore presenza di una CAD. Il presente studio si \ue8 proposto di indagare il possibile ruolo delle CAD nel corso dello sviluppo e maturazione del frutto di pesco in relazione alle caratteristiche di tessitura e/o alla diversa colorazione della polpa. Analisi bioinformatiche ci hanno permesso di determinare che in pesco le cad costituiscono una vasta famiglia multigenicacon oltre 60 sequenze putative; di queste circa 40, con elevata identit\ue0 di sequenza, sono allocate sullo scaffold 6. Sono stati quindi valutati i livelli di attivit\ue0 CAD in vitro in frutti di pesco con diversa durezza e/o colorazione e durante le diverse fasi dello sviluppo. In generale, questi sono risultati trascurabili nel mesocarpo dei frutti Melting Flesh a polpa gialla e pi\uf9 alti nei frutti con elevata durezza o di colore rosso. I risultati sono stati confrontati con quelli relativi alla valutazione della attivit\ue0 in gel dopo elettroforesi nativa e Western blot con anticorpi anti CAD di F. x ananassa, costituiti contro un putativo ortologo delle CAD dello scaffold 6 di pesca. Non sempre vi \ue8 concordanza tra attivit\ue0 CAD in gel e segnale anticorpale, suggerendo la possibilit\ue0 che altre isoforme di CAD siano attive nei tessuti e nelle fasi di sviluppo analizzate. Tale ipotesi \ue8 confermata anche da analisi preliminari RT-PCR che hanno permesso di rilevare come i trascritti di diverse CAD siano simultaneamente presenti all\u2019interno dello stesso tessuto in proporzione variabile a seconda dello stadio di sviluppo e in relazione al genotipo. Ulteriori analisi sono in corso per delucidare il complesso quadro che va delineando i possibili ruoli fisiologici delle differenti CAD

The maize pentatricopeptide repeat gene empty pericarp4 (emp4) is required for proper cellular development in vegetative tissues

The empty pericarp4 (emp4) gene encodes a mitochondrion-targeted pentatricopeptide repeat (ppr) protein that is involved in the regulation of mitochondrial gene expression and is required for seed development. In homozygous mutant emp4-1 kernels the endosperm is drastically reduced and the embryo is retarded in its development and unable to germinate. With the aim of investigating the role of emp4 during post-germinative development, homozygous mutant seedlings were obtained by cultivation of excised immature embryos on a synthetic medium. In the mutants both germination frequency as well as the proportion of seedlings reaching the first and second leaf stages were reduced. The anatomy of the leaf blades and the root cortex was not affected by the mutation, however severe alterations such as the presence of empty cells or cells containing poorly organized organelles, were observed. Moreover both mitochondria and chloroplast functionality was impaired in the mutants. Our hypothesis is that mitochondrial impairment, the primary effect of the mutation, causes secondary effects on the development of other cellular organelles. Ultra-structural features of mutant leaf blade mesophyll cells are reminiscent of cells undergoing senescence. Interestingly, both structural and functional damage was less severe in seedlings grown in total darkness compared with those exposed to light, thus suggesting that the effects of the mutation are enhanced by the presence of light

Genes interaction in maize endosperm development

In this report we present the results of a complementation test involving nine emp (empty pericarp) mutants of maize representing single gene mutants, isolated as independent events. These mutants are embryo lethal and drastically reduced in their endosperm size. As to the endosperm, they can be subdivided into two major subgroups: one including those with a flat appearance of the kernel and smooth pericarp and another one with a wrinkled pericarp. In all mutants, the analysis of longitudinal sections of mature seeds reveals absence of morphogenesis in the embryo proper, an observation the correlates with the failure of these mutants to germinate. By crossing inter-se plants heterozygous for the nine emp mutants we identified those non complementing (i.e. allelic) and those complementing (i.e. not allelic) in the F1 generation. Most results in the F1 were concordant to those obtained in the F2 generation with the exception of three cases where the F1 results suggest allelism (i.e. one gene) whereas those in the F2 suggest segregation of two genes. These intriguing results seem to suggest an interaction between different emp mutants, attributable to a phenomenon that is often referred to as second site non-complementation (SSNC). SSNC can be explained by assuming interaction between two different mutant proteins leading to a toxic product or that the mutant form of one protein sequesters the wild-type form of the other protein into an inactive complex. In some cases of 9 to 7 segregation in the F2 generation we recovered the two phenotypes (emp flat and emp wrinkled) with values fitting the 9:4:3 ratio suggesting the presence of an epistatic interactions between the two mutants. For example, in the case of the cross between emp4 and emp*-9475 the scoring of F2 ears revealed a segregation ratio of 9:4:3 (wt: emp wrinkled: emp flat) in which we can assume that emp*-9475, that has a wrinkled phenotype, is epistatic to emp4, that has a flat phenotype. However the data need to be increased to demonstrate that the hypothesis postulated is correct. Another important point was the finding that in some ears segregating for a single emp mutant in different genetic backgrounds (A636, W23 and Mo17) some mutant seeds were identified exhibiting a more abundant endosperm tissue and occasionally an embryonic axis. A low percentage of these seeds germinate. This observation could be explained in two ways. The first implies that emp mutants uncover a cryptic variability and the second possibility is that the improved endosperm of the mutant is the result of an interaction between the mutant and a second factor originally present in the inbred line. The results of crosses made to test for a segregation of a modifier have allowed us to identify a dominant and a recessive modifier. If these results are confirmed, emp mutants could be used as a tool for the detection of genetic factors enhancing the amount of endosperm in the maize kernel to be exploited in breeding programs

A mutational approach for the detection of genetic factors affecting seed size in maize

Genes influencing seed size. The designation emp (empty pericarp) refers to a group of defective kernel mutants that exhibit a drastic reduction in endosperm tissue production. They allow the isolation of genes controlling seed development and affecting seed size. Nine independently isolated emp mutants have been analyzed in this study and in all cases longitudinal sections of mature seeds revealed the absence of morphogenesis in the embryo proper, an observation that correlates with their failure to germinate. Complementation tests with the nine emp mutants, crossed inter se in all pairwise combinations, identified complementing and non-complementing pairs in the F1 progenies. Data were then validated in the F2/F3 generations. Mutant chromosomal location was also established. Overall our study has identified two novel emp genes and a novel allele at the previously identified emp4 gene. The introgression of single emp mutants in a different genetic background revealed the existence of a cryptic genetic variation (CGV) recognizable as a variable increase in the endosperm tissue. The unmasking of CGV by introducing single mutants in different genetic backgrounds is the result of the interaction of the emp mutants with a suppressor that has no obvious phenotype of its own and is present in the genetic background of the inbred lines into which the emp mutants were transferred. On the basis of these results, emp mutants could be used as tools for the detection of genetic factors that enhance the amount of endosperm tissue in the maize kernel and which could thus become valuable targets to exploit in future breeding programs