Minisymposium 19: Membrane Transport

Abs # 33002: ACA9, a calmodulin-activated calcium pump involved in pollen growth and fertilization

Presenter:	Harper, Jeffrey F, Harper@scripps.edu
Authors	Harper, Jeffrey F (A)   Schiøtt, Morten  (B)   Romanowsky, Shawn  (A)   Palmgren, Michael  (B)
Affiliations:	(A): The Scripps Research Institute (B): The Royal Vetrinary and Agricultural University, Copenhagen, Denmark

Calcium signals play an important role in plant growth and development, including key aspects of pollen tip growth and fertilization. The dynamics of a calcium signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). Efflux pathways include antiporters and pumps. In Arabidopsis there are 14 calcium pumps from two distinct families. The ACAs (autoinhibited calcium ATPase, 10 isoforms) are characterized by an N-terminal autoinhibitor. At least some members have been shown to be activated by calmodulin. ACAs have been so far been found in the ER, tonoplast and plasma membrane. The ECAs (ER-type calcium ATPase, 4 isoforms) have only been found in the ER. To investigate the biological functions of the plant calcium pumps we have isolated T-DNA disruptions in all 14 of the Arabidopsis calcium pumps. No single pump is essential under laboratory conditions, as shown by the ability to recover viable homozygous plants for each of the 14 gene disruptions. Nevertheless, a single disruption of ACA9 (autoinhibited calcium ATPase, isoform 9) displays a partial sterility phenotype. Homozygous aca9 mutants (three independent alleles) all showed around an 85% reduction in seed-set (about 8 seeds per silique). This appears to result from a pollen defect, as supported by the observation that wild-type pollen can produce full seed-set when used to fertilize a homozygous aca9. A pollen phenotype is consistent with evidence for pollen-specific expression of ACA9, as revealed by promoter::GUS-fusions. Mutant pollen have at least two defects. First, they show reduced growth potentials in vitro and in vivo. This results in a reduced frequency of pollen tubes reaching ovules in the distal end of the pistil. Second, mutant pollen displayed a block in fertilization in more than 50% of the cases in which pollen tubes reached the ovule micropyle. These blocked fertilization events provide an explanation for the partial seed set reduction (25% decrease) observed as a semi-dominant phenotype in heterozygotes (i.e. competition by mutant pollen). Preliminary evidence suggests that ACA9 is localized to the plasma membrane, as indicated by an ACA9-GFP fusion and complementation by a known PM calcium pump (ACA8). This is the first genetic evidence for the role of a specific calcium transporter in pollen growth and fertilization.