Disruption of Apyrases Inhibits Pollen Germination in Arabidopsis

Abstract

In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive ␤-glucuronidase staining in the pollen of apyrase promoter:␤-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.Pollen germination represents a short, yet very critical event in a series of steps leading to the fertilization of the ovule. Mature pollen is shed from the anther as desiccated grains and rehydrates upon contact with the stigma. During hydration, the pollen shape changes from elongated to round, and Ca 2ϩ enters the pollen grain, leading to rearrangement of the cytoskeleton and establishment of a Ca 2ϩ gradient (Heslop-Harrison and Heslop-Harrison, 1992a, 1992b; for schematic illustration, see Johnson and McCormick, 2001). Germination follows rapidly, usually within minutes in Arabidopsis (Pruitt and Hü lskamp, 1994), and is visually manifested by the emergence of a pollen tube. Although pollen germination in vivo is a tightly regulated process dependent on multiple cues from the stigma (Johnson and Preuss, 2002), pollen grains will germinate in vitro in aqueous medium minimally containing Suc, boric acid, and Ca 2ϩ (Taylor and Hepler, 1997). Few genes have been identified so far that are specific for the germination stage (for reviews, see Franklin-Tong, 2002; Johnson and Preuss, 2002). Here, we introduce the enzyme apyrase as an essential player in this early gametophytic phase.Apyrases hydrolyze nucleoside tri-and diphosphates and are highly active and most likely ubiquitous, because they have been found in all pro-and eukaryotic organisms examined for their presence. In animals, these enzymes have been shown to play important regulatory roles in the mediation of signaling events, for example the quenching of the neurotransmitter ATP (Plesner, 1995;Komoszynski and Wojtczak, 1996).The function of plant apyrases has been studied most intensively in legumes, namely Dolichos biflorus, wild soybean (Glycine soja), Medicago truncatula, and pea (Pisum sativum). In wild soybean and D. biflorus, two apyrases each we...

Apyrases (Nucleoside Triphosphate-Diphosphohydrolases) Play a Key Role in Growth Control in Arabidopsis

“…The amino acid sequences of APY1 and APY2 are 87% identical and these two apyrases at least partially complement each other's function (Steinebrunner et al, 2003). However, the tissue expression patterns of these two apyrases are different, at least in roots, so we cannot assume their subcellular distribution is identical.…”

“…However, DKO progeny could not be produced easily because DKO pollen cannot germinate (Steinebrunner et al, 2003). Therefore, a complementation strategy with an inducible promoter was chosen: Double-heterozygous plants (apy1/1; apy2/1) for a T-DNA insertion in the APY1 (apy1) and APY2 (apy2) genes were transformed with cDNA for either APY1 or APY2, each under the dexamethasone (DEX)-inducible promoter (Aoyama and Chua, 1997).…”

“…. The apy1 and apy2 mutants were isolated previously (Steinebrunner et al, 2003). For root and hypocotyl growth assays, seeds were sown on the surface of solidified Murashige and Skoog medium.…”

“…They are found in all eukaryotes and are far more efficient in removing phosphates from NTP/ nucleoside diphosphate than other phosphatases. They are characterized by conserved motifs (Handa and Guidotti, 1996) and by their relative insensitivity to specific inhibitors of P-type, F-type, and V-type ATPases and to most inhibitors of alkaline and acid phosphatases (Zimmermann, 2001;Steinebrunner et al, 2003).…”

“…Overexpression of APY2 lowers the sensitivity of Arabidopsis leaves to applied ATP , consistent with the hypothesis that it can function as an ectoapyrase. An initial genetic investigation of the role of APY1 and APY2 revealed that they were needed for pollen germination (Steinebrunner et al, 2003).…”