Genetic mechanisms of maize development: from gametophyte to flowers

Zea mays L. is one of the world’s most agronomically important crop. The understanding of the molecular basis of inflorescences architecture and seed development may be useful for agronomic purposes. The major goal of this research is to investigate different aspect of maize development to shed light on the genetic mechanisms involved in the formation of maize inflorescences as well as seed development. In the first part of my thesis, the mechanisms regulating inflorescences development have been investigated by studying a new barren mutant, barren inflorescence173 (bif173). The recessive mutant bif173 is affected in the formation of axillary meristems, showing defects in inflorescences development, such as a reduction in the number of spikelets and branches in the tassel and smaller and more disorganized ears. The phenotype of this mutant is not fully penetrant and its severity seems to be related to temperature or light changes. Also, we demonstrated that bif173, like other barren mutants, is involved in auxin biology and may play a role in auxin signaling. In order to identify the gene responsible of bif173 mutation, a RNA-seq analysis was carried out to closely examine a mapping region previously identified and one SNP present only in bif173 mutant transcripts was found. This SNP represents a non-synonymous mutation in the coding region of the gene GRMZM2G038401, causing a change of a very conserved amino acid in the encoded protein. This gene encodes a metalloprotease, homologous to the FtsH ATP- dependent metalloproteases, a conserved family of membrane- bound proteases. The ubiquitous localization of the GRMZM2G038401 transcripts seems to be consistent with the numerous functions of these proteases. As evidence that GRMZM2G038401 gene is a good candidate for bif173 mutation is the fact that the SNP found in the RNA-seq reads was not present in teosinte and other maize inbred lines, suggesting that it is not a polymorphism due to the genetic variability among maize background. In order to confirm that GRMZM2G038401 is the gene responsible for bif173 mutation, plants homozygous for a transposon insertion are currently growing and if the phenotype resembles the bif173 mutant phenotype, this gene will be confirmed as the causative gene. This finding will shed light on the molecular mechanisms regulating inflorescences development in maize and will increase our knowledge in auxin biology. In the second part of my thesis, genetic mechanisms acting in seed development have been investigated, particularly focusing on gametogenesis and embryogenesis. In A. thaliana, DME is a gene encoding a DNA glycosylase/lyase, active in the central cell of the female gametophyte before fertilization. The role of this enzyme is essential for the viability of the seed, in fact, acting as a demethylase, it activates the expression of maternal alleles, establishing imprinting in the endosperm. Here, two DME homologues in maize were identified: ZmDME1 and ZmDME2. The proteins encoded by these genes showed a high homology with A. thaliana DME and a conserved protein structure characteristic of the DME family. A phylogenetic analysis also suggested that these proteins have a common evolutionary origin. The expression of these genes was found in different stages of gametogenesis, previously identified through a morphological analysis. ZmDME1 and ZmDME2 showed a different expression pattern compared to A. thaliana DME, i.e. the expression was not only found in the mature gametophyte containing the central cell, but also in the embryo and endosperm and in all the vegetative tissues tested. Furthermore, the localization of the expression of ZmDME1 and ZmDME2 in the mature gametophyte was detected not only in the central cell but also in the other cells of the embryo sac and in the nucellus. In A. thaliana dme mutants produce non viable seeds, with enlarged endosperm and aborted embryos. A functional analysis using Zmdme1 mutant plants revealed no defects in vegetative and reproductive phases, producing all normal-shaped seeds. A morphological analysis of these mutants showed that gametogenesis and embryogenesis occur normally. Nevertheless, further analyses are needed to verify the function of these genes. Even if the lack of DME orthologues in monocots has been previously hypothesized, recent findings suggest that a similar mechanism of DNA demethylation may take place in monocot gametophyte. Thus, we discuss about the possibility that ZmDME1 and ZmDME2 may be responsible of active demethylation in maize gametophyte, allowing the proper development of embryo and endosperm.