The eukaryotic genome is duplicated exactly once per cell division cycle. A strategy that limits every replication origin to a single initiation event is tightly regulated by a multiprotein complex, which involves at least 20 protein factors. A key player in this regulation is the evolutionary conserved hexameric MCM2-7 complex. From maize (Zea mays) zygotes, we have cloned MCM6 and characterized this essential gene in more detail. Shortly after fertilization, expression of ZmMCM6 is strongly induced. During progression of zygote and proembryo development, ZmMCM6 transcript amounts decrease and are low in vegetative tissues, where expression is restricted to tissues containing proliferating cells. The highest protein amounts are detectable about 6 to 20 d after fertilization in developing kernels. Subcellular localization studies revealed that MCM6 protein shuttles between cytoplasm and nucleoplasm in a cell cycle-dependent manner. ZmMCM6 is taken up by the nucleus during G1 phase and the highest protein levels were observed during late G1/S phase. ZmMCM6 is excluded from the nucleus during late S, G2, and mitosis. Transgenic maize was generated to overexpress and down-regulate ZmMCM6. Plants displaying minor antisense transcript amounts were reduced in size and did not develop cobs to maturity. Down-regulation of ZmMCM6 gene activity seems also to affect pollen development because antisense transgenes could not be propagated via pollen to wild-type plants. In summary, the transgenic data indicate that MCM6 is essential for both vegetative as well as reproductive growth and development in plants.

Imprinting refers to the epigenetic regulation of gene expression that is dependent upon gene inheritance from the maternal or paternal parent. Previously, we have identified two maize homologs of the single Arabidopsis Polycomb Group gene FIE. Here, we report on the expression pattern of these genes in individual gametes before and after fertilization, and on the role of DNA methylation in determining the maternal expression of the Fie1 gene. We found that Fie1 is neither expressed in the sperm, egg cell nor central cell before fertilization. Activation of the Fie1 maternal allele occurs around two days after pollination (DAP) in the primary endosperm and peaks at 10-11 DAP coinciding with endosperm transition from mitotic division to endoreduplication. In contrast, Fie2 is expressed in the egg cell and more intensively in the central cell similar to Arabidopsis FIE, which strongly supports the hypothesis that it functions as a repressor of endosperm development before fertilization. Using MSRE-PCR and bisulfite sequencing, we could show that the methylated inactive state is the default status of Fie1 in most tissues. In the endosperm the paternal Fie1 allele remains methylated and silent, but the maternal allele appears hypomethylated and active, explaining mono-allelic expression of Fie1 in the endosperm. Taking together, these data demonstrate that the regulation of Fie1 imprinting in maize is different from Arabidopsis and that Fie1 is likely to have acquired important novel functions for endosperm development.

Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter- and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50–100 μm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Reversible post-translational modification of numerous proteins by small ubiquitin-related modifiers (SUMOs) represents a major regulatory process in various eukaryotic cellular and developmental processes. To study the role of sumoylation during female gametophyte (FG) development in maize, we identified Zea mays genes encoding SUMO (ZmSUMO1a and ZmSUMO1b) and a diSUMO-like protein called ZmDSUL that contains two head-to-tail SUMO-like domains. Whereas ZmSUMO1a and ZmSUMO1b are almost ubiquitously expressed, ZmDSUL transcripts were detected exclusively in the egg apparatus and zygote. ZmDSUL was selected for detailed studies. ZmDSUL is processed close to the C-terminus, generating a dimeric protein that is similar to animal FAT10 and ISG15, which contain two ubiquitin-like domains. Whereas GFP fused to the ZmDSUL N-terminus was located in the cytoplasm and predominately in the nucleoplasm of some transiently transformed maize suspension cells, C-terminal GFP fusions exclusively accumulated at the nuclear surface. GFP or ZmDSUL-GFP under control of the ZmDSUL promoter first displayed GFP signals in the micropylar-most position of the FG at stage 5/6, when migration of polar nuclei and cellularization occurs. Mature FGs displayed GFP signals exclusively in the egg cell, but the strongest signals were observed shortly after fertilization and disappeared during the first asymmetric zygotic division. RNAi silencing of ZmDSUL showed that it is required for FG viability. Moreover, nuclei segregation and positioning defects occurred at stage FG 5 after mitotic nuclear divisions were completed. In summary, we report a diSUMO-like protein that appears to be essential for nuclei segregation and positioning, the prerequisite for cell specification during FG maturation.