Stocky1, a Novel Gene Involved in Maize Seedling Development and Cuticle Integrity.

The cuticle is the plant's outermost layer that covers the surfaces of aerial parts. This structure is composed of a variety of aliphatic molecules and is well-known for its protective role against biotic and abiotic stresses in plants. Mutants with a permeable cuticle show developmental defects such as organ fusions and altered seed germination and viability. In this study, we identified a novel maize mutant, stocky1, with unique features: lethal at the seedling stage, and showing a severely dwarfed phenotype, due to a defective cuticle. For the first time, the mutant was tentatively mapped to chromosome 5, bin 5.04. The mutant phenotype investigated in this work has the potential to contribute to the elucidation of the role of the cuticle during plant development. The possibility of controlling this trait is of relevance in the context of climate change, as it may contribute to tolerance to abiotic stresses.

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

KEY MESSAGE: 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.

A mutational approach to the study of seed development in maize.

The maize seed comprises two major compartments, the embryo and the endosperm, both originating from the double fertilization event. The embryogenetic process allows the formation of a well-differentiated embryonic axis, surrounded by a single massive cotyledon, the scutellum. The mature endosperm constitutes the bulk of the seed and comprises specific regions containing reserve proteins, complex carbohydrates, and oils. To gain more insight into molecular events that underlie seed development, three monogenic mutants were characterized, referred to as emp (empty pericarp) on the basis of their extreme endosperm reduction, first recognizable at about 12 d after pollination. Their histological analysis reveals a partial development of the endosperm domains as well as loss of adhesion between pedicel tissues and the basal transfer layer. In the endosperm, programmed cell death (PCD) is delayed. The embryo appears retarded in its growth, but not impaired in its morphogenesis. The mutants can be rescued by culturing immature embryos, even though the seedlings appear retarded in their growth. The analysis of seeds with discordant embryo-endosperm phenotype (mutant embryo, normal endosperm and vice-versa), obtained using B-A translocations, suggests that emp expression in the embryo is necessary, but not sufficient, for proper seed development. In all three mutants the picture emerging is one of a general delay in processes related to growth, as a result of a mutation affecting endosperm development as a primary event.

empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize.

The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35-amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4-green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.

Genetic analysis as a tool to investigate the molecular mechanisms underlying seed development in maize.

BACKGROUND: In angiosperms the seed is the outcome of double fertilization, a process leading to the formation of the embryo and the endosperm. The development of the two seed compartments goes through three main phases: polarization, differentiation of the main tissues and organs and maturation. SCOPE: This review focuses on the maize kernel as a model system for developmental and genetic studies of seed development in angiosperms. An overview of what is known about the genetic and molecular aspects underlying embryo and endosperm formation and maturation is presented. The role played by embryonic meristems in laying down the plant architecture is discussed. The acquisition of the different endosperm domains are presented together with the use of molecular markers available for the detection of these domains. Finally the role of programmed cell death in embryo and endosperm development is considered. CONCLUSIONS: The sequence of events occurring in the developing maize seed appears to be strictly regulated. Proper seed development requires the co-ordinated expression of embryo and endosperm genes and relies on the interaction between the two seed components and between the seed and the maternal tissues. Mutant analysis is instrumental in unravelling the genetic control underlying the formation of each compartment as well as the molecular signals interplaying between the two compartments.

Programmed cell death during embryogenesis in maize.

Programmed cell death (PCD) in plants is considered an integral part of development. Evidence of DNA fragmentation, occurring at specific sites and times during embryo formation in maize (Zea mays L.), was obtained using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labelling (TUNEL) and by genomic DNA ladder detection. During the crucial period of elaboration of the primary shoot and root axis (14-20 d after pollination), TUNEL-positive nuclei are present in the scutellum, coleoptile, root cap and principally in the suspensor. Additional evidence of a form of programmed cell death occurring in these tissues comes from the detection of a DNA ladder. Upon completion of the differentiation process, all embryonic cells are TUNEL-negative, indicating that possible programmed cell death events during maize embryogenesis are confined to structures or organs that do not contribute to the adult plant body.

Mutations in two independent genes lead to suppression of the shoot apical meristem in maize.

The shoot apical meristem (SAM), initially formed during embryogenesis, gives rise to the aboveground portion of the maize (Zea mays) plant. The shootless phenotype (sml) described here is caused by disruption of SAM formation due to the synergistic interaction of mutations at two genetic loci. Seedlings must be homozygous for both sml (shootmeristemless), and the unlinked dgr (distorted growth) loci for a SAM-less phenotype to occur. Seedlings mutant only for sml are impaired in their morphogenesis to different extents, whereas the dgr mutation alone does not have a recognisable phenotype. Thus, dgr can be envisaged as being a dominant modifier of sml and the 12 (normal):3 (distorted growth):1 (shoot meristemless) segregation observed in the F(2) of the double heterozygote is the result of the interaction between the sml and dgr genes. Other segregation patterns were also observed in the F(2), suggesting instability of the dgr gene. Efforts to rescue mutant embryos by growth on media enriched with hormones have been unsuccessful so far. However, mutant roots grow normally on medium supplemented with kinetin at a concentration that suppresses wild-type root elongation, suggesting possible involvement of the mutant in the reception or transduction of the kinetin signal or transport of the hormone. The shootless mutant appears to be a valuable tool with which to investigate the organization of the shoot meristem in monocots as well as a means to assay the origins and relationships between organs such as the scutellum, the coleoptile, and leaves that are initiated during the embryogenic process.