Gene expression profiles of cold-stored and fresh pollen to investigate pollen germination and growth.

In lily (Lilium longiflorum cv. Avita) pollen cold-stored (-20 degrees C) for 2 months, typical in vitro germination/growth was delayed by about 1 h 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. Cold-stored and fresh pollen grains were used to investigate the regulatory mechanism of pollen germination and tube growth in terms of both total protein profile and gene expression. Total protein profiles of cold-stored pollen differed qualitatively and quantitatively from fresh pollen. Actinomycin D significantly inhibited 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. 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 h. Because of different sensitivities to cold storage and actinomycin D, the transcripts were divided into three groups according to their possible roles in pollen germination and tube growth. Several cDNAs encoding novel proteins showed pollen-specific expression patterns and may participate in drought 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.

Molecular characterization of a novel senescence-associated gene SPA15 induced during leaf senescence in sweet potato.

The structure and expression of a novel senescence-associated gene (SPA15) of sweet potato were characterized. The protein coding region of the gene consists of 13 exons encoding 420 amino acids. Apparent homologues of this sweet potato gene are found in a variety of dicot and monocot plants, but not in animals or microorganisms. Examination of the expression patterns of the SPA15 gene in sweet potato reveals that the transcripts of SPA15 are specifically induced in the senescing leaves, and the temporal profile of SPA15 protein accumulation is correlated with that of SPA15 transcripts. Studies on the distribution of SPA15 homologue in rice plants also indicate that SPA15 homologue is up-regulated specifically in senescing rice leaves. Treatment of detached sweet potato leaves with phytohormones including ethylene, methyl jasmonate, salicylic acid and abscisic acid resulted in a high-level induction of SPA15. Immunoelectron microscopic analysis demonstrates that SPA15 is specifically associated with the cell wall. The potential role for SPA15 during leaf senescence is discussed.

Molecular characterization of a senescence-associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet potato.

The structure and expression of a senescence-associated gene (SPG31) encoding a cysteine proteinase precursor of sweet potato have been characterized. The coding region of the gene consists of two exons encoding an enzyme precursor of 341 amino acids with conserved catalytic amino acids of papain. Examination of the expression patterns of the SPG31 gene in sweet potato by Northern blot analyses reveals that the transcripts of SPG31 are specifically induced in the senescing leaves but not in other organs. The differential accumulation of the mature SPG31 protein in the senescing leaves was further identified by two-dimensional electrophoresis of leaf proteins and N-terminal sequencing. This result suggests the important role played by SPG31 in proteolysis and nitrogen remobilization during the leaf senescence process. Furthermore, treatment of mature green leaves with ethylene for 3 d resulted in a high-level induction of SPG31 transcripts. Ethylene-regulated expression of SPG31 is consistent with the presence of a number of putative ethylene-responsive elements in the 899-bp SPG31 promoter region.

Cloning and characterization of leaf senescence up-regulated genes in sweet potato.

Genes that are expressed during leaf senescence in sweet potato (Ipomoea batatas, cv. Tainong 57) were identified by the isolation of cDNA fragments with the mRNA differential display method. Eight senescence-associated cDNA clones for mRNAs differentially expressed during leaf senescence were obtained and characterized. Northern blot analysis indicated that all these clones represented genes that are up-regulated during natural leaf senescence. Among them, five cDNA clones have been obtained in full length by screening a senescing leaf cDNA library or by performing rapid amplification of cDNA ends. DNA and protein database searches revealed that clones SPA15 and SPC9 encode proteins of unknown function. The other six clones SPG31, SPC20, SPG27, SPC25, SPC15 and SPC1 showed significant sequence homology to known genes encoding a cysteine proteinase, isocitrate lyase, S-adenosylmethionine decarboxylase, cysteine proteinase inhibitor and metallothionein-like type I protein. The gene expression patterns represented by SPG31, SPG27 and SPA15 were found to be highly specific in senescing leaves. The corresponding transcripts for SPG31, SPG27 and SPA15 were below detectable levels in other organs such as flowers, stems, roots and tubers. The possible physiological roles of these gene products in the leaf senescence process are discussed.