High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Diphtheria toxin-mediated cell ablation in developing pollen: Vegetative cell ablation blocks generative cell migration

Summary The technique of genetic cell ablation involves the targeted expression of a cell autonomous cytotoxic protein under the control of cell-specific regulatory sequences. This technique allows the investigation of cell-cell interactions by inducing selective death in a precisely controlled and cell autonomous manner. Here, targeted vegetative cell-specific ablation was used to examine the role of the vegetative cell (VC) in controlling generative cell (GC) behaviour and differentiation during pollen development. The tomatolat 52 late-pollen promoter, which has been shown to be activated specifically in the nascent VC immediately following pollen mitosis I (PMI), was used to direct expression of the cytotoxic diphtheria toxin A chain (DTA) in both transient expression assays using microprojectile bombardment and in transgenic tobacco plants. Transient expression of DTA linked to thelat 52 promoter (lot 52-DTA) in pollen dramatically reduced the expression of a co-transfected reporter gene fusion, demonstrating the cytotoxicity of DTA to pollen. Genetic and phenotypic analysis oflat 52-DTA transformants demonstrated that DTA expression led to a pollen-lethal phenotype, recognisable as small acytoplasmic pollen grains at anthesis, which affected 50% of the pollen population in single locus transformants. Detailed cytological analysis using confocal laser scanning microscopy and vital staining using fluorescein diacetate (FDA), showed that the first sign of cell ablation during pollen development was a loss of vital staining of the VC immediately following PMI. In contrast, the GC retained viability for up to several days following VC ablation, but progressively lost viability in the absence of a functional VC. Of particular interest was the observation that in the absence of VC function the generative cell (GC) failed to undergo normal migration away from the pollen grain wall into the VC cytoplasm. These results directly demonstrate the dependence of the GC on VC cell functions and highlight the importance of VC-GC interactions in controlling GC migration.Abbreviations CaMV cauliflower mosaic virus - nos nopaline synthase - DTA diptheria toxin A chain - lat late anther tomato - VC vegetative cell - GC generative cell - PGM pollen germination medium - EtBr ethidium bromide - FDA fluorescein diacetate - FCR fluorochrome reaction - DAPI 4,6-diamidino-2-phenylindole     

Characterization and functional analysis of a pollen-specific gene st901 in Solanum tuberosum

A pollen-specific gene, sb401, which was isolated from a cDNA library of in vitro geminated pollen of the diploid potato species Solanum berthaultii, belongs to the class of genes expressed late during pollen development. Using sb401 as a probe, a pollen-specific gene st901 was isolated from the genomic library of a potato species Solanum tuberosum cv. Desiree. Sequencing and RT-PCR analysis showed that the st901 genomic gene is 2,889 bp long, contains three exons and two introns, and encodes a putative polypeptide of 217 residues. The predicted protein sequence contains four imperfect repeated motifs of V–V–E–K–K–N/E–E; the core sequence of the repeats (K–K–N/E–E) resembles a microtubule-binding domain of the microtubule-associated protein MAP1B from mouse. The examination of a promoter–reporter construct in transgenic potato plants revealed that the st901 is expressed exclusively in mature pollen grains, which is consistent with the results of Northern blot and RT-PCR. For analysis of the function of st901, transgenic plants harboring antisense copies of st901 cDNA driven by a native st901 promoter were generated. Suppression of st901 gene in potato resulted in aberrant pollen at maturation and pollen viability of transgenic plants ranged from 4.4 to 14.8%, while that of control plants were more than 90%. These results strongly suggest that st901 has an essential role in pollen development.The st901 gene sequence has been deposited in GenBank under accession number AY526087. Accession number for SB401 is X95984.1