Search

F-box protein genes family is one of the largest gene families in plants, with almost 700 predicted genes in the model plant Arabidopsis. F-box proteins are key components of the ubiquitin proteasome system that allows targeted protein degradation. Transcriptome analyses indicate that half of these F-box protein genes are found expressed in microspore and/or pollen, i.e., during male gametogenesis. To assess the role of F-box protein genes during this crucial developmental step, we selected 34 F-box protein genes recorded as highly and specifically expressed in pollen and isolated corresponding insertion mutants. We checked the expression level of each selected gene by RT-PCR and confirmed pollen expression for 25 genes, but specific expression for only 10 of the 34 F-box protein genes. In addition, we tested the expression level of selected F-box protein genes in 24 mutant lines and showed that 11 of them were null mutants. Transmission analysis of the mutations to the progeny showed that none of the single mutations was gametophytic lethal. These unaffected transmission efficiencies suggested leaky mutations or functional redundancy among F-box protein genes. Cytological observation of the gametophytes in the mutants confirmed these results. Combinations of mutations in F-box protein genes from the same subfamily did not lead to transmission defect either, further highlighting functional redundancy and/or a high proportion of pseudogenes among these F-box protein genes.

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in Arabidopsis thaliana; RPT5a and RPT5b. We showed previously that RPT5a and RPT5b are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), RPT5b does not complement the effect of a strong rpt5a mutation in the male gametophyte, and only partially complements rpt5a mutation in the sporophyte. RPT5bCol-0 and RPT5bWs-4 differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene., Results: By exploiting natural variation at RPT5b we determined that the SNP located in RPT5b intron seven, rather than the promoter SNP, is the sole basis of this lack of redundancy. In Ws-4 this SNP is predicted to create a new splicing branchpoint sequence that induces a partial mis-splicing of the pre-mRNA, leading to the introduction of a Premature Termination Codon. We characterized 5 accessions carrying this A-to-T substitution in intron seven and observed a complete correlation between this SNP and both a 10 to 20% level of the RPT5b pre-mRNA mis-splicing and the lack of ability to complement an rpt5a mutant phenotype., Conclusion: The accession-dependent unequal redundancy between RPT5a and RPT5b genes illustrates an example of evolutionary drifting between duplicated genes through alternative splicing.

We investigated the role of the ubiquitin proteasome system (UPS), which allows proteins to be selectively degraded, during gametophyte development in Arabidopsis thaliana. Three mutant alleles altering the UPS were isolated in the Wassilewskija (Ws) accession: they affect the Regulatory Particle 5a (RPT5a) gene, which (along with RPT5b) encodes one of the six AAA-ATPases of the proteasome regulatory particle. In the heterozygous state, all three mutant alleles displayed 50% pollen lethality, suggesting that RPT5a is essential for male gametophyte development. However, a fourth mutant in the Columbia (Col) accession did not display such a phenotype because the RPT5b Col allele complements the rpt5a defect in the male gametophyte, whereas the RPT5b Ws allele does not. Double rpt5a rpt5b mutants showed a complete male and female gametophyte lethal phenotype in a Col background, indicating that RPT5 subunits are essential for both gametophytic phases. Mitotic divisions were affected in double mutant gametophytes correlating with an absence of the proteasome-dependent cyclinA3 degradation. Finally, we show that RPT5b expression is highly increased when proteasome functioning is defective, allowing complementation of the rpt5a mutation. In conclusion, RPT5 subunits are not only essential for both male and female gametophyte development but also display accession-dependent redundancy and are crucial in cell cycle progression.

The organization and dynamics of the plant endomembrane system require both universal and plant-specific molecules and compartments. The latter, despite the growing wealth of information, remains poorly understood. From the study of an Arabidopsis thaliana male gametophytic mutant, it was possible to isolate a gene named POKY POLLEN TUBE (POK) essential for pollen tube tip growth. The similarity between the predicted POK protein sequence and yeast Vps52p, a subunit from the GARP/VFT complex which is involved in the docking of vesicles from the prevacuolar compartment to the Golgi apparatus, suggested that the POK protein plays a role in plant membrane trafficking. Genetic analysis of Arabidopsis mutants affecting AtVPS53 or AtVPS54 genes which encode putative POK partners shows a transmission defect through the male gametophyte for all lines, which is similar to the pok mutant. Using a combination of biochemical approaches and specific antiserum it has been demonstrated that the POK protein is present in phylogenetically divergent plant species, associated with membranes and belongs to a high molecular weight complex. Combination of immunolocalization studies and pharmacological approaches in different plant cells revealed that the POK protein associates with Golgi and post-Golgi compartments. The role of POK in post-Golgi endomembrane trafficking and as a member of a putative plant GARP/VFT complex is discussed.

In higher plants, pollen tubes and root hairs share an ancient growth process named tip growth. We have isolated three allelic Arabidopsis mutant lines showing kinky-shaped pollen tubes and, when homozygous, showing shorter and thicker root hairs. The ultrastructure of pollen tubes in these kinky pollen (kip) mutants is similar to that of the wild type; however, time-lapse studies suggest that aberrant pollen tube shape is caused by periodic growth arrests alternated with phases of tube axis reorientation. The KIP gene encodes a protein of 2587 amino acids that is predicted to be targeted to the secretory pathway. KIP mRNA was detected in all organs investigated but was most abundant in pollen and roots. KIP has putative homologues in many eukaryotes, including mammals and yeast, and is similar to the Arabidopsis SABRE gene, whose mutation causes a dwarf phenotype. The phenotype of the kip/sab double mutant suggests related functions for both genes, however, the KIP protein is mostly required for tip-growth.

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 KanR:KanS segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.