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 °C) and arabinose concentration at 2%, during the induction period, increase the amount of protein produced by the bacteria.