40S Ribosome Biogenesis Co-Factors Are Essential for Gametophyte and Embryo Development

Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants.     

The Arabidopsis phosphatidylinositol 3-kinase is important for pollen development

Phosphatidylinositol 3-kinase has been reported to be important for normal plant growth. To characterize the role of the enzyme further, we attempted to isolate Arabidopsis (Arabidopsis thaliana) plants that do not express the gene, but we could not recover homozygous mutant plants. The progeny of VPS34/vps34 heterozygous plants, harboring a T-DNA insertion, showed a segregation ratio of 1:1:0 for wild-type, heterozygous, and homozygous mutant plants, indicating a gametophytic defect. Genetic transmission analysis showed that the abnormal segregation ratio was due to failure to transmit the mutant allele through the male gametophyte. Microscopic observation revealed that 2-fold higher proportions of pollen grains in heterozygous plants than wild-type plants were dead or showed reduced numbers of nuclei. Many mature pollen grains from the heterozygous plants contained large vacuoles even until the mature pollen stage, whereas pollen from wild-type plants contained many small vacuoles beginning from the vacuolated pollen stage, which indicated that vacuoles in many of the heterozygous mutant pollen did not undergo normal fission after the first mitotic division. Taken together, our results suggest that phosphatidylinositol 3-kinase is essential for vacuole reorganization and nuclear division during pollen development.     

拟南芥温度诱导脂质运载蛋白TIL1参与雌配子体发育

雌配子体的正常发育是种子形成的前提条件之一,拟南芥温度诱导的脂质运载蛋白编码基因TIL1突变使胚珠败育,结实率下降明显。基因表达分析表明T-DNA插入使得TIL1基因敲除,突变体TIL1基因功能缺失;互交实验、Alexander染色、花粉离体培养和胚珠透明实验结果表明till-1突变体雄配子体发育正常、雌配子体胚囊发育有缺陷;通过遗传互补实验证明外源克隆的TIL1基因能恢复突变体的败育表型,并确定了TIL1基因主要在胚珠的胚囊中表达。实验结果表明TIL1基因参与了植物雌配子体发育这一重要的生理过程。     

T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics

T-DNA insertion mutants have become a valuable resource for studies of gene function in Arabidopsis. In the course of both forward and reverse genetic projects, we have identified novel interchromosomal rearrangements in two Arabidopsis T-DNA insertion lines. Both rearrangements were unilateral translocations associated with the left borders of T-DNA inserts that exhibited normal Mendelian segregation. In one study, we characterized the embryo-defective88 mutation. Although emb88 had been mapped to chromosome I, molecular analysis of DNA adjacent to the T-DNA left border revealed sequence from chromosome V. Simple sequence length polymorphism mapping of the T-DNA insertion demonstrated that a >40-kbp region of chromosome V had inserted with the T-DNA into the emb88 locus on chromosome I. A similar scenario was observed with a prospective T-DNA knockout allele of the LIGHT-REGULATED RECEPTOR PROTEIN KINASE (LRRPK) gene. Whereas wild-type LRRPK is on lower chromosome IV, mapping of the T-DNA localized the disrupted LRRPK allele to chromosome V. In both these cases, the sequence of a single T-DNA-flanking region did not provide an accurate picture of DNA disruption because flanking sequences had duplicated and inserted, with the T-DNA, into other chromosomal locations. Our results indicate that T-DNA insertion lines--even those that exhibit straightforward genetic behavior--may contain an unexpectedly high frequency of rearrangements. Such duplication/translocations can interfere with reverse genetic analyses and provide misleading information about the molecular basis of mutant phenotypes. Simple mapping and polymerase chain reaction methods for detecting such rearrangements should be included as a standard step in T-DNA mutant analysis.     

Arabidopsis glucan synthase-like 10 functions in male gametogenesis

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.