The late pollen-specific actins in angiosperms.

Muthugapatti K. Kandasamy, Elizabeth C. McKinney andRichard B. Meagher*Department of Genetics, Life Science Building, Universityof Georgia, Athens, GA 30602, USASummaryThe actin gene family of Arabidopsis has eight functionalgenes that are grouped into two ancient classes,vegetative and reproductive, and into ﬁve subclassesbased on their phylogeny and mRNA expressionpatterns. Progress in deciphering the functionalsigniﬁcance of this diversity is hindered by the lack oftools that can distinguish the highly conservedsubclasses of actin proteins at the biochemical andcellular level. In order to address the functional diversityof actin isovariants, we have used Arabidopsisrecombinant actins as immunogens and producedseveral new anti-actin monoclonal antibodies. One ofthem, MAb45a, speciﬁcally recognizes two closelyrelated reproductive subclasses of actins. Onimmunoblots, MAb45a reacts strongly with actinsexpressed in mature pollen but not with actins in otherArabidopsis tissues. Moreover, immunocytochemicalstudies show that this antibody can distinguish actinﬁlaments in pollen tubes from those in most vegetativetissues. Peptide competition analyses demonstrate thatasparagine at position 79 (Asn79) within an otherwiseconserved sequence is essential for MAb45a speciﬁcity.Actins with the Asn79 epitope are also expressed in themature pollen from diverse angiosperms and Ephedrabut not from lower gymnosperms, suggesting that thisepitope arose in an ancestor common to angiospermsand advanced gymnosperms more than 220 millionyears ago. During late pollen development in angio-sperms there is a switch in expression of actins fromvegetative to predominantly reproductive subclasses,perhaps to fulﬁl the unique functions of pollen infertilization.IntroductionActin is a major, dynamic component of the plantcytoskeleton. It provides a structural framework for cells,andinadditionmediatesanumber ofimportantsubcellularprocesses in plants. Cytochemical and physiological stu-dies have shown that actin ﬁlaments are involved in cellshape and division plane determination, cell wall deposi-tion, transport, organelle movement, nuclear positioning,cytoplasmic streaming, tip growth and polysome distribu-tion (reviewed in Meagher, 1991; Meagher etal., 1999a;Staiger and Lloyd, 1991). Thus, the actin cytoskeleton isessential for cell growth and differentiation and for thedevelopment of organs and the plant as a whole.The functional diversity of actins is paralleled by thediversity within plant actin gene families which comprisedozens or even hundreds of actin sequences (Meagher andWilliamson, 1994). The eight expressed actin genes inArabidopsis represent the best-characterized plant actinfamily. Quantitative evolutionary studies and extensiveanalyses of steady-state RNA levels and transgenic plantsexpressing actin gene–reporter fusions in Arabidopsisreveal two major classes of actin genes: a vegetative classand a reproductive class. The vegetative class is composedof two subclasses (1: ACT2 and ACT8; 2: ACT7) and boththese subclasses are expressed predominantly in leaves,stems, roots, petals and sepals. The reproductive class iscomposed of three subclasses (3: ACT11; 4: ACT1 andACT3; 5: ACT4 and ACT12), all of which are stronglyexpressed in pollen. ACT11 is also expressed in all ovuleand embryonic tissues (McDowell etal., 1996; Meagheretal., 1999b). The two actins in the same subclasses, suchas ACT1 and ACT3 of subclass 4 (Figure1) differ only by asingle amino acid and shared a common ancestralsequence more than 30 million years ago (McDowell etal.,1996). The reproductive class actins differ from thevegetative class actins by 4–6.8% in amino acid sequence,suggesting that they have not shared common ancestry forat least 350–400 million years. The divergence times forthese two actin classes and most of the actin subclassespre-date the origin of angiosperms. We hypothesize thatthese actin subclasses have been conserved duringvascular plant evolution because they exhibit advanta-geous expression patterns or encode actin isovariants withunique functions. Although it has been shown in animalsthat different isovariants of actin have some specializedfunctions (reviewed in Fyrberg etal., 1998; Herman, 1993;Rubenstein, 1990), nothing is known about the functionalsigniﬁcance of the presence of multiple plant actinisovariants (McLean etal., 1990). We also do not knowwhether the transcriptional expression of reporters andmRNA steady-state levels accurately reﬂects protein