In general, there are two major research themes in our laboratory: genes/proteins and potential autophagic machinery involved in plant reproduction.
GENES/PROTEINS INVOLVED IN PLANT REPRODUCTION
(A) Genes/proteins involved in pollen germination and tube growth.
The efficient germination of pollen tube is critical for successful double fertilization in plant sexual reproduction. Lily (Lilium longiflorum) pollen grains stored at -20¢XC for 2 months, the pollen germination/growth was significantly delayed by about 1 hr compared with fresh pollen. We hypothesized that some proteins and mRNAs stored in mature pollen were degraded during storage periods and that re-synthesis of them was essential to resume normal germination and growth (Wang et al., 2004). Cold-stored and fresh pollen grains were used to investigate the regulatory mechanism of pollen germination and tube growth in both total protein profile and gene expression. Total protein profiles of cold-stored pollen differed qualitatively and quantitatively from fresh pollen. Actinomycin D significantly retarded both germination and tube growth of cold-stored pollen and later tube growth of fresh pollen but had no effect on fresh pollen germination and early tube growth. Above results suggest both protein and mRNA levels in pollen grain are significantly affected during low temperature storage. Suppression subtractive hybridization screening revealed 99 cDNAs enriched in fresh mature pollen, and 22 were selected for further characterization. Most of these 22 cDNAs gradually disappeared during cold storage, but full recovery was achieved by incubating the cold-stored pollen in culture medium for 2 hr. Several cDNAs encoding novel proteins showed pollen-specific expression patterns and may participate in drought 44 45 Annual Report 2005 tolerance (an Na+/H+ antiporter), endomembrane trafficking (DnaJ), division of the generative cell (Sgt1), pollen wall precursor uptake from stylar exudate (an Na+/myoinositol symporter), and chemotropism of the pollen tube (peptide transporter) during pollination. Their functions in pollination are under investigating.
Figure 1. Microarray scanning and tree view graph analyses of the cDNAs enriched in the in vivo-grown pollen tubes.
(A) More than 5600 cDNA clones enriched in the in vivo-grown pollen tubes were obtained after suppression subtraction hybridization and the PCR products of these clones were spotted on slides for array chip preparation. The mRNAs purified from in vivo-grown 24 hr and in vivo-grown 12 or 24 hr pollen tubes labeled with Cy5-dUTP and Cy3-dUTP, respectively, were used as probes for array chips hybridization. Pseudo color obtained after microarray analyses indicated that those cDNAs were either up-regulated in the vivo pollen tubes (red spots), in the in vitro pollen tube (green spots) or expressed cDNAs (yellow spots). (B) Microarray results were analyzed by GeneSpring software for gene expression pattern presented as tree view graph.
Pollen tube is a good model to study cell-cell interaction and polar growth. For the pollen expressed genes, almost all of them found in the literature were obtained from in vitro or semi-in vivo condition and very little information about those of in vivo-grown pollen tubes has been characterized. In lily, we have the unique advantage of being able to extract in vivo-grown pollen tubes from the style for comparison to in vitro-grown pollen tubes. Suppression subtractive hybridization screening was performed to obtain transcripts enriched and/or specifically expressed in the in vivo-grown pollen. Approximately 5000 clones enriched in the in vivo-grown pollen tubes were obtained from the sub-library, used for array chips preparation, and subjected for microarray analyses (Figure 1). 140 cDNAs were obtained and sequenced and their expression patterns were further examined by RT-PCR. Sixteen genes were not pre-accumulated in pollen grains but expressed after pollen germination or tube elongation. Among them, 12 genes mainly expressed in the in vivo-grown pollen tubes and 4 genes showed developmental expression during pollen germination. The spatial expression analyses showed that 11 transcripts were only detected in the in vivo-grown pollen tubes and pistil. The results presented here greatly broaden our current knowledge about the regulation of gene expression during pollination and imply that the stylar/stigma exudates may stimulate gene expression in the in vivo-grown pollen tubes. Further characterization of the potential functions of these transcripts by transgenic plant study is in progress.
POTENTIAL AUTOPHAGIC MACHINERY INVOLVED IN POLLEN DEVELOPMENT
Figure 2. Autophagosome-like compartments enclosed amyloplasts and lipid body during lily pollen germination.
In vitro-germinated 60 minutes lily pollen grains embedded in Spurr's resin, were prepared and subjected to ultrastructural investigation. A. During lily pollen germination, the tubular ER (arrows) enveloped two different kinds of organelles, the amyloplasts (a) and lipid bodies (1), to form autophagosome-like compartments. B. Higer magnification micrograph showed a typical well differentiated amyloplast found in germinating pollen surrounded by one layer of tubular ERs (marked by arrows) to form the autophagosome-like structure. C and D are two independent pictures to show that the "antenna-like" margins of starch granules progressively invade this enclosed inner membrane system. E. It was often to find that the lipid body was also surrounded by one layer of tubular ERs (arrows) to form the autophagosome-like structure which was going to completely seal the enclosed lipid body and left an open ands indicated by arrowheads. a: amyloplast, g: Golgi apparatus, l: lipid body, m: mitochondria, s: starch granule,v: vacuole, Bars=1 μm (A), 0.2 μm (B), 0.1 μm (C), 0.5 μm (D,E).
Autophagy is an intracellular process of bulk protein degradation which occurs in most eukaryotic organisms. Most of our current understanding of autophagy arises from research performed in yeast. During the starvation, yeast cell will initiate autophagy pathway to degrade and to recycle cytoplasmic compartments for survival purposes. The process begins with the formation of the autophagosome which is a double-membrane vesicle composed of a portion of the cytosol. This vesicle will continue to fuse with vacuole which contains proteases to breakdown the autophagosome thus complete the autophagy process. Lily pollen germinated in culture medium for different time intervals a lot of amyloplasts containing various-sized starch granules started accumulation. Our ultrastructural observation revealed that it was usually observed these amyloplasts were enveloped by a double membrane structure with morphology similar to tubular endoplasmic reticulum to form the autophagosome-like compartments (Figure 2). Not only starch granules, some membranous structures similar to mitochondria were often found inside these compartments. In addition to starch granules, some membrane debris and lipid bodies, but not amyloplast, were found in the vacuolar lumen. The possible autophagic machinery may be involved in the utilization and degradation of reserves, such as starch granules, and vacuole ontogeny
during pollen germination.
Wang, M. L., Hsu, C. M., Chang, L. C., Wang, C. S., Su, T.-H., Huang, J. Y. J., Jiang, L., and Jauh, G. Y. 2004. Gene expression profiles of cold-stored and fresh pollen to investigate pollen germination and growth. Plant Cell Physiol. 45: 1519-1528.
Moriyasu, Y., Hattori, M., Jauh, G. Y., and Rogers, J. C. 2003. Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process. Plant Cell Physiol. 44: 795-802.
